IC-NRLF 


BACTERIOLOGICAL 


TECHNOLOGY 


- 


LIBRARY 

OF  THK 

UNIVERSITY  OF  CALIFORNIA 

OIPT  OR 


Received 
Accession  No. 


•    Class  No.   BIOLOGY 


^  v. 


BACTERIOLOGICAL  TECHNOLOGY 


FOR   PHYSICIANS 


WITH    SEVENTY-TWO   FIGURES   IN    THE   TEXT 


BY 


DR.   C.  J.   SALOMONSEN 


Authorized  Translation  from  the  Second  Revised  Danish  Edition 

BY 

WILLIAM    TRELEASE 


- 
s 

~-\  - 


NEW  YORK 

WILLIAM  WOOD  &  COMPANY 
1890 


BIOLOGY 
R* 


COPYRIGHT,  1890, 

BY 

WILLIAM  WOOD  &  COMPANY 


AUTHOR'S   PREFACE. 


THE  first  edition  of  this  text-book  was  issued  in  1885,  as  a 
reprint  from  the  "  Nordiskt  Medicinskt  Arkiv."  The  increased 
size  of  this  new  edition  is  partly  due  to  the  addition  of  an  in- 
troduction to  the  microscopic  investigation  of  bacteria;  but 
the  active  development  of  bacteriology  in  the  last  few  years 
has  also  necessitated  a  re-elaboration  and  extension  of  all  of 
the  chapters. 

In  the  work,  I  have  had  two  objects  in  view:  the  prepara- 
tion of  an  outline  adapted  to  bacteriological  courses  for  physi- 
cians and  veterinary  surgeons,  and  a  guide  for  those  who  are 
obliged  to  take  up  the  subject  at  home  without  the  assistance 
of  an  instructor,  yet  wish  to  carry  out  for  themselves  the 
fundamental  experiments  which  are  most  important  for  path- 
ology and  hygiene.  I  have,  therefore,  not  attempted  to  give 
an  exhaustive  presentation  of  the  entire  technology  of  the 
subject,  but  rather  to  describe  the  simplest  and  most  easily 
managed  apparatus  and  methods,  so  that  the  equipment  of  a 
home  laboratory  need  not  appear  too  expensive;  while  I  have 
tried  to  describe  these  in  a  sufficiently  elementary  and  de- 
tailed manner. 

Those  who  wish  a  more  comprehensive  treatise,  are  re- 
ferred to  the  last  edition  of  Hueppe's  "  Methoden  der  Bakte- 
rienf orschung  "  (Wiesbaden,  1889). 

C.  J.  S. 


TRANSLATOR'S  PREFACE. 


So  many  works  on  bacteriology  are  now  accessible,  that  a- 
new  one  must  possess  decided  merit  to  justify  its  appearance. 
The  scope  of  the  present  little  volume  is  such  as  to  satisfy  me 
that  it  should  fill  a  place  as  yet  vacant ;  and  the  careful  treat- 
ment of  the  subject  gives  rise  to  the  hope  that  it  may  find  a 
larger  field  of  usefulness  in  the  English  language  than  could 
be  possible  while  it  was  confined  to  the  less-used  Danish. 

The  physician  who  wishes  to  read  a  well-written  account 
of  the  more  important  pathogenic  bacteria,  is  further  referred 
to  Fraenkel's  "  Bakterienkunde."  The  fullest  and  most  re- 
liable attempt  at  a  bacteriological  flora,  is  to  be  found  in 
Fluegge's  "  Ferment e  und  Mikroparasiten,"  which  contains 
much  additional  information. 

W.  T. 


BACTERIOLOGICAL  TECHNOLOGY. 


CHAPTER  I. 


STERILIZATION. 

BACTERIOLOGICAL  technology  must  in  the  first  place  put 
means  in  our  hands  for  separating-  the  different  kinds  of  bac- 
teria from  one  another,  and  cultivating-  each  species  in  a  state 
of  complete  purity.  Pure  cultivation  is  a  necessary  condition 
for  a  trustworthy  study  of  the  morphology  and  physiology 
of  these  microscopic  organisms.  Unless  one  has  this  con- 
stantly in  mind  during  his  work,  he  is  certain  to  end  in  sad 
confusion;  and  the  short  history  of  bacteriology  is  only  too 
full  of  worthless  investigations  and  wrong-  results,  which  are 
to  be  attributed  to  the  untrustworthiness  of  the  methods  em- 
ployed. 

It  is  well  known  that  the  pure  cultivation  of  a  single  kind 
of  bacteria  is  surrounded  by  quite  peculiar  difficulties,  due  to 
the  extreme  smallness,  the  large  numbers,  and  the  extraordi- 
nary distribution,  one  might  almost  say  omnipresence,  of  bac- 
teria. Everything  which  is  allowed  to  stand  uncovered  in  the 
air  for  a  short  time  is  likely  to  receive  spores  capable  of  g-er- 
mination,  which  fall  on  its  surface  with  the  dust.  Every  con- 
tact with  hands  or  clothing-  is  attended  with  the  same  dang-er. 
We  must  learn  first  of  all  to  meet  and  control  this  accidental 
and  invisible  source  of  contamination,  for  it  is  self-evident  that 
all  vessels,  instruments,  fluids,  etc.,  which  are  to  be  used  in 
cultures  must  be  clean,  not  only  in  the  sense  in  which  chemists 
use  the  word,  but  also  in  the  bacteriological  sense,  or  sterile, 
that  is,  free  from  living  germs. 

Heat  is  the  most  generally  used  means  of  sterilization,  but 
1 


2  Bacteriological    Technology. 

it  must  be  used  in  different  ways,  and  of  different  degrees, 
according*  to  the  nature  of  the  material  to  be  sterilized,  a 
platinum  needle,  for  example,  is  best  sterilized  by  heating  it 
to  incandescence  directly  in  the  flame;  but  a  knife  blade,  or  a 
pair  of  scissors,  can  clearly  not  be  heated  to  this  point.  These 
can  only  be  "  flamed/'  that  is,  drawn  slowly  back  and  forth 
through  the  flame,  until  one  is  sure  that  all  of  their  surface 
has  been  heated  to  at  least  200°  C.,  so  that  any  germs  have 
been  burned. 

But  it  is  only  exceptionally  that  glowing  or  flaming  can  be 
employed.  If  one  has  to  deal  with  glass-ware,  metallic  arti- 
cles, cotton,  paper,  etc.,  these  are  most  frequently  sterilized  in 
air  heated  to  about  150°  C.  In  the  preparation  of  most  fluid 
and  solid  culture-media,  one  can  most  rapidly  and  surely 
reach  the  desired  end  by  the  help  of  a  Papin  digester,  an 
"  autoclave,"  in  which  the  sterilization  is  effected  by  steam 
under  pressure,  at  110  to  120°  C.;  or  one  contents  himself  with 
using  the  boiling  temperature,  either  by  directly  cooking  the 
substance,  or  by  heating  it  to  100°  C.,  or  about  that  point,  by 
the  aid  of  streaming  steam.  Occasionally,  however,  one  may 
have  to  use  a  temperature  lower  than  this  (60  to  70°  C.),  to 
kill  germs,  for  instance  where  serum  is  used.  In  some  cases 
filtration  and  disinfecting  solutions  are  used  as  means  of  ster- 
ilizing. 

The  entire  collection  of  instruments  needed  for  sterilizing, 
the  apparatus  and  culture-media  to  be  used  in  the  investiga- 
tions that  we  shall  describe,  are,  in  addition  to  a  common 
Bunsen  gas  burner  (or  alcohol  lamp),  the  following : 

1.  A  sterilizing  oven  in  which  articles  made  of  glass  and 
metal,  cotton,  paper,  etc.,  are  heated  to  about  150°  C.,  by  means 
of  hot  air. 

For  this  purpose  one  uses  in  the  laboratory  a  double-walled 
sheet-iron  box  30  cm.  high,  35  cm.  broad,  and  23  cm.  deep,  with 
a  tube  in  the  top  for  inserting  a  thermometer,  a  door  in  the 
side,  and  with  a  loose  middle  piece  in  the  bottom  against 
which  the  large  gas  jet  constantly  plays,  and  which  conse- 
quently will  burn  through  after  long  use,  when  it  is  only 
necessary  to  put  in  a  new  piece  of  iron. 

One  can  usually  get  along  with  less  expense  by  making  a 
serviceable  sterilizing  oven  (Figs.  1,  2)  of  one  of  the  common 
cracker  boxes  (about  24  cm.  high,  22  cm.  wide,  and  24  cm. 


Bacteriological    Technology. 


deep)  to  be  obtained  cheaply  of  any  grocer.  In  the  cover  a 
round  hole  is  to  be  cut  in  which  a  bored  cork  is  fitted  to  carry 
a  thermometer,  registering-  to  200°  C.  For  ventilation,  a  small 
hole  is  punched  in  each  of  the  four  side-walls  close  to  the  top 
and  bottom.  In  the  box  is  set  a  four-sided  piece  of  strong 
sheet-iron,  with  the  sides  bent  down  so  as  to  hold  it  about 
2  cm.  from  the  bottom,  so  that  the  objects  to  be  sterilized  will 
not  come  into  immediate  contact  with  the  latter.  The  box  is 
inclosed  in  a  substance  which  is  a  poor  conductor  of  heat,  felt 
being  usually  chosen.  One  piece  covers  the  lid,  and  is  perfor- 


FlG.   1. 


FIG.  2. 


FIGS.  1  and  2.— Oven  for  sterilization  at  150°  C.,  formed  of  a  cracker  box  surrounded 
with  felt  and  provided  with  thermometer  and  support  of  iron  netting. 

ated  for  the  passage  of  the  cork  and  thermometer.  Another 
piece  is  chosen  long  enough  to  be  wrapped  around  the  four 
sides  of  the  box  and  wide  enough  to  cover  the  upper  three 
quarters  of  these,  excepting-  a  small  space  where  the  air-holes 
occur.  The  lowermost  quarter  is  best  left  uncovered  to  avoid 
scorching-  or  burning  the  felt.  This  strip  of  felt  is  fastened 
simply  by  tying  around  it  tightly  a  few  pieces  of  cord  or  wire. 
The  box  is  set  on  one  or  two  of  the  common  iron  tripods,  such 
as  are  used  in  chemical  laboratories,  and  is  heated  by  one  or 
more  gas  burners.  Between  these  and  the  bottom  of  the  box, 
a  loose  piece  of  sheet-tin  can  be  placed  to  prevent  burning 


4  Bacteriological    Technology. 

through  the  bottom.  If  gas  is  not  at  hand,  a'small  oil  stove 
with  one  or  two  burners  can  be  used. 

In  comparison  with  the  larger  and  more  expensive  ovens 
provided  with  ventilators  and  t  her  mo -regulator,  this  primitive 
apparatus  offers  some  inconveniences,  but  these  do  not  count 
for  much  in  comparison  with  its  perfect  usefulness  and  ex- 
treme cheapness. 

Care  must  be  taken  to  guard  the  sterilized  objects  from 
fresh  contamination  when  they  are  taken  out  of  the  sterilizer. 
Vessels  which  are  guarded  from  the  penetration  of  germs,  by 
cotton  plugs  or  otherwise,  when  it  is  not  especially  important 
to  keep  the  outside  free  from  germs  (e.g.,  flasks  and  test-tubes) 
can  simply  be  taken  from  the  oven  after  they  have  been  heated 
to  150°  C.  for  half  an  hour  or  an  hour,  and  set  aside  without  any 
other  precaution  than  covering  or  wrapping  them  up  in  paper 
to  prevent  them  from  becoming  very  dusty.  But  objects 
which  must  be  perfectly  sterilized,  such  as  watch-glasses, 
slides,  large  glass  plates,  etc.,  must  be  carefully  wrapped  in  a 
couple  of  thicknesses  of  thin,  firm  paper  before  they  are  put  in 
the  oven.  Germs  from  the  air  cannot  pass  through  this,  and 
the  objects  can  thus  be  kept  satisfactorily  sterile  for  a  long 
time.  It  is  well  to  wrap  each  object  in  several  layers  of  thin 
paper,  and  then  to  wrap  these  in  larger  parcels. 

Paper  and  cotton  should  be  kept  as  far  as  possible  from 
the  bottom  of  the  oven  to  prevent  them  from  becoming 
charred  by  long  heating.  Paper  and  cotton  (but  not  absorb- 
ent cotton)  assume  a  yellowish-brown  color  after  being  kept  at 
from  140°  to  150°  C.  for  a  long  time,  and  this  discoloration  con- 
sequently shows  that  the  heat  in  the  sterilizing  oven  has  been 
sufficient  even  without  the  testimony  of  a  thermometer. 

2.  The  Steam  Sterilizer  of  Koch. — Sterilization  at  the  boil- 
ing temperature  can  evidently  be  effected  by  cooking  in  water, 
but  since  this  method  is  not  only  inapplicable  in  many  cases, 
but  always  more  laborious  and  unreliable  than  sterilization 
by  streaming  steam  as  introduced  by  Koch,  the  latter  is  now 
the  principal  method  used  when  it  is  desired  to  sterilize  at 
100°  C. 

It  will  be  remembered  that  two  advantages  are  gained  by 
this:  the  heat  penetrates  rapidly  into  the  objects  to  be  steril- 
ized, and  steam  of  this  temperature  (99°  to  100°  C.  according 
to  altitude)  sterilizes  much  more  rapidly  than  atmospheric  air 


Bacteriological    Technology. 


5 


of  the   same  or   a   much  higher  temperature,   probably   for 
chemical  reasons. 

The  partly  diagrammatic  illustration  shows  the  Koch  steam 
apparatus  of  a  simple  and  cheap  form.     It  consists  of  a  tin 


FIG.  3. 


FIG.  4. 


FIGS.  3  and  4.— Koch  cylinder  in  its  simplest  form,  with  two  tin  shelves  and  pails.  A, 
Hook  for  suspending  long  objects  ;  #,  tin  pail  with  sieve-bottom,  containing  plugged  test- 
tubes  ;  C,  tin  cylinder  for  elongating  the  apparatus. 

cylinder  50  cm.  high  and  17  cm.  in  diameter,  and  is  provided 
with  a  small  glass  tube  (a)  fastened  by  rubber  tubing  in  which 
the  height  of  the  water  can  be  seen  from  the  outside.  The 
absolute  size,  as  well  as  the  proportion  between  the  height 


6  Bacteriological  'Technology. 

and  diameter,  can  naturally  be  varied  within  wide  limits  and 
the  distance  between  the  shelves  is  to  be  adapted  to  the 
height  of  the  apparatus,  etc.,  that  are  to  be  most  frequently 
sterilized.  The  bottom,  which  is  to  be  exposed  to  the  action 
of  the  flame,  is  preferably  made  of  somewhat  thicker  tin  than 
the  sides.  The  top  of  the  apparatus  is  closed  by  a  conical  lid 
(b)  the  centre  of  which  is  pierced  by  a  short  tube  (c)  for  the 
reception  of  a  cork  and  thermometer.  The  lid  and  the  side 
walls,  to  within  9  cm.  of  the  bottom,  are  usually  covered  with 
felt,  though  this  is  not  absolutely  necessary.  Within  the 
cylinder,  at  a  distance  of  14  and  30  cm.  from  the  bottom,  are 
placed  projecting1  ledges,  on  which  can  be  placed  two  loosely 
fitting  shelves  of  heavy  tin  perforated  with  large  holes  (del). 
These  serve  to  support  tin  pails  (ee)  about  12  cm.  high  and  14 
cm.  in  diameter,  the  bottom  of  which  is  best  made  of  wide- 
meshed  galvanized  iron  netting  (Fig.  4,  B).  These  hold  the 
objects  to  be  sterilized,  e.g.,  test-tubes  plugged  with  cotton 
in  the  figure.  When  it  is  to  be  used,  the  apparatus  is  placed 
on  a  tripod  (/)  of  stout  strap-iron.  The  pails  are  taken  out 
and  the  cylinder  is  filled  with  water  to  a  depth  of  about  10 
cm.  The  pails  with  their  contents  having  been  set  in  and  the 
lid  with  its  thermometer  put  in  place,  the  water  is  heated  to 
the  boiling  point.  After  some  time  steam  will  escape  freely 
around  the  loosely-fitting  lid,  and  the  thermometer  will  also 
show  about  100°  C.,  after  which  the  objects  are  left  in  this 
streaming  steam  for  a  longer  or  shorter  time  (as  a  rule, 
twenty  minutes),  the  time  being  counted  from  the  moment 
when  the  thermometer  indicates  100°  C.  (or  the  steam  begins 
to  escape  freely).  Most  objects  which  are  to  be  sterilized  by 
steam  can  be  set  on  the  tin  shelves  without  difficulty.  Longer 
objects  can  be  hung  in  the  apparatus  after  the  pails  and 
shelves  have  been  removed.  This  is  most  easily  done  by  using 
a  piece  of  stout  brass  wire,  bent  and  brought  into  the  opening 
of  the  lid  (Fig.  4,  A). 

The  apparatus  can  be  lengthened  when  this  is  necessary 
by  using  a  tin  cylinder,  with  a  projecting  collar  near  its  base 
(Fig.  4,  D C)  to  be  inserted  between  the  lid  and  the  top  of  the 
cylinder.  A  stop-cock,  near  the  bottom,  for  drawing  off  the 
water,  increases  the  expense  of  the  apparatus  somewhat,  but 
adds  to  its  convenience.  Moreover,  one  can  easily  form  such  a 
steam  sterilizer  from  a  common  kitchen  pot.  It  is  only  neces- 


Bacteriological    Technology.  j 

sary  to  fit  to  it  a  tin  cylinder  open  at  the  bottom,  and  fur- 
nished with  a  lid  and  felt  covering1,  but  it  is  essential  to  pro- 
vide this  cylinder  with  a  projecting-  collar  at  a  couple  of 
centimetres  from  the  bottom,  by  which  it  rests  on  the  edge  of 
the  pot;  and  it  is  best  to  provide  it  with  a  couple  of  rings,  by 
which  the  cylinder  may  be  tied  to  the  ears  of  the  pot. 

A  word  of  explanation  should  be  offered  for  the  omission 
of  the  Papin  digester  in  speaking-  of  necessary  apparatus  for 
sterilization.  There  are,  indeed,  bacillus  spores  which  are 
able  to  survive  a  cooking  for  several  hours  in  water  or  free 
steam,  while  they  are  killed  after  a  few  moments'  heating-  to 
120°  C.  in  the  digester,  consequently  this  is  the  only  apparatus 
in  which  one  can  rapidly  and  surely  effect  sterilization  by  a 
single  attempt.  Still  it  is  a  very  expensive  piece  of  apparatus, 
which  is  the  only  reason  that  it  has  been  omitted.  Koch's 
steam  cylinder,  on  the  other  hand,  is  very  simple,  cheap,  and 
efficient  when  it  is  used  with  care.  '  As  a  rule  the  spores  of 
bacilli  which  always  get  into  our  culture  media,  from  water, 
the  air,  etc.,  are  not  so  resistant  as  those  which  have  been 
mentioned.  Generally,  streaming1  steam  at  100°  C.  will  kill 
them,  and  experiments  from  a  large  number  of  bacteriological 
laboratories  have  long  since"  shown  the  adequac}^  of  Koch's 
apparatus.  However,  in  the  preparation  of  his  culture  appar- 
atus one  must  always  have  in  mind  that  some  very  resistant 
form  may  have  escaped  destruction  in  the  steam,  and  must 
make  sure  of  absolute  freedom  from  germs  in  his  culture 
media  by: 

a.  Cleanliness  in  the  preparation.     It  is  not  sufficient  to 
trust  to  the  final  sterilization  of  the  materials,  but  at  every 
step  of  the  preparation  one  should  work  cleanly,  to  lessen  the 
chances  of  resistant  g-erms  entering. 

b.  Discontinuous  Heating. — This  method  was  first  applied 
by  Tyndall  in  1877  for  the  sterilization  of  hay  infusion,  which 
is  known  to  contain  some  of  the  most  resistant  bacillus  spores, 
the  extraordinary  vitality  of  which  renders  it  impossible  to 
sterilize  this  substance  even  by  cooking-  it  for  some  minutes 
(c/.  Chapter  IV.,  Section  2).     Tyndall  reached  his  method  of 
sterilizing-  by  the  following  train  of  thought :     When  a  hay 
infusion  has  been  boiled  for  some  minutes  all  of  the  rod -shaped 
bacilli,  unable  to  endure  this  treatment,  are  dead,  while  the 
spores  of  bacilli  (more  exactly,  of  some  of  them)  are  still  alive. 


Bacteriological    Technology. 

If  now  all  of  these  surviving-  spores  are  given  time  to  germi- 
nate and  the  infusion  is  again  cooked,  it  will  be  completely 
sterilized,  because,  when  it  is  heated  it  contains  no  spores  but 
only  rods.  However,  as  one  cannot  be  sure  that  all  spores 
have  germinated  at  the  same  time,  this  process  must  usually 
be  repeated  several  times.  Hence  it  is  recommended  to  submit 
the  filled  culture  apparatus  to  steaming  twice,  with  a  day  in- 
tervening. This  is  especially  important  for  those  culture 
media  which,  like  gelatin,  bear  only  a  short  cooking. 

c.  Waiting  and  Watching. — For  greater  certainty,  the 
materials  treated  in  this  way  are  allowed  to  stand  for  some 
time  before  they  are  used,  so  that  one  may  be  sure  that  they 
contain  nothing  capable  of  germination.  If  they  are  placed 
in  the  thermostat  at  about  30°  C.,  a  delay  of  a  couple  of  days 
may  be  looked  on  as  sufficient. 

3.  Water  Bath  for  Sterilizing  at  Temperatures  below  the 
Boiling  Point. — Various  fluids  will  not  bear  being  sterilized  at 
100°  C.,  because  at  so  high  a  temperature  they  undergo  chem- 
ical changes  which  it  is  wished  to  avoid  for  one  reason  or 
another.  In  such  cases  the  short  exposure  to  a  high  temper- 
ature can  be  replaced  by  long  maintenance  at  a  lower  tem- 
perature, until  no  germ  capable  of  growth  remains  in  the 
fluids.  This  method  was  discovered  by  Pasteur,  and  by  his 
advice  applied  on  a  large  scale  in  the  fabrication  of  wine 
("  Pasteurization  ")  to  secure  sterilization  of  the  wines  without 
at  the  same  time  injuring  their  quality.  In  what  follows  we 
shall  make  use  of  the  preparation  of  blood  serum  according  to 
Koch's  directions.  Rather  complicated  and  expensive  appar- 
atus is  often  used  for  this,  but  one  can  get  along  very  well 
with  a  simple  cylindrical  water-bath  made  of  tin,  such  as 
Koch  himself  used  in  his  first  preparations.  The  tin  receptacle 
(Fig.  5)  is  22  cm.  high,  and  13  cm.  in  diameter.  At  the  top  it 
is  provided  with  a  collar  1.5  cm.  broad.  A  piece  of  fine-meshed 
flexible  galvanized  iron  netting  (a)  serves  as  a  lid,  and  is  fast- 
ened by  bending  its  edges  under  the  collar.  The  temperature 
of  the  water  is  read  off  on  the  thermometer  (6)  which  projects 
through  a  hole  in  the  lid.  The  bulb  of  the  thermometer  is 
held  up  several  centimetres  above  the  bottom  of  the  cylinder, 
by  another  loose  piece  of  wire  cloth  (c)  resting  upon  its  edges, 
which  are  bent  down;  when  the  water  has  reached  a  suitable 
temperature,  usually  60°  to  70°  C.,  it  is  easy  to  hold  it  at  about 


Bacteriological    Technology. 


the  same  point  for  a  long  time,  e.g.,  a  couple  of  hours,  "by  the 
use  of  a  small  gas  or  alcohol  flame.  Though  a  range  of  a  few 
degrees  can  usually  be  allowed,  it  is  best  to  keep  the  tempera- 
ture under  observation.  Further  directions  for  the  prepara- 
tion of  serum  are  given  later. 

4.  Porcelain  Filters. — Even  a  slight  heating  often  causes 
in  the  culture  fluid  chemical  changes  which  it  may  be  import- 
ant to  avoid.  In  such  cases  the  germs  have  been  removed  by 
filtration  through  burnt  clay  (Klebs),  plaster  (Pasteur  and 
Miquel),  porcelain  (Chamberland),  or  sheet  asbestos  (Hesse), 


FIG.  6. 

Fio.  5.— Water-bath  for  Sterilization  at  Lower  Temperatures. 

FIG.  G.  —  A,  Chamberland  Filter  (X  1/6);  B,  bulb-pipette  for  collecting  the  contents  of 
the  filter;  E,  small  porcelain  filter  (x  1  /6);  F,  reservoir,  which  can  also  be  used  as  a  cul- 
ture-vessel; C  and  D  are  explained  in  the  text. 

which  keep  back  all  of  the  cells  contained  in  the  fluid.  As 
filtration  plays  a  prominent  part  in  the  separation  of  bacteria 
from  their  soluble  products,  we  shall  here  dwell  somewhat 
more  fully  on  the  use  of 

Ckamberland's  Filter.— This  consists  of  a  hollow  porcelain 
cylinder  (Fig.  6  A\  which  is  closed  at  the  bottom  and  at  the 
upper  end  provided  with  a  funnel-shaped  glazed  end-piece, 
over  which  a  rubber  tube  can  be  pushed.  The  filter  is  im- 
mersed in  the  fluid  to  be  sterilized  and  the  upper  (open)  end  is 
connected  with  some  kind  of  aspirator.  If  the  fluid  to  be  fil- 


10 


Bacteriological    Technology. 


tered  is  in  too  great  quantity  to  be  contained  in  the  cavity  of 
the  filter,  another  receptacle  is  inserted  between  the  aspirator 
and  filter.  An  ordinary  wash-bottle  (Fig-.  ?)  can  be  used  for 
this  purpose;  and  when  the  fluid  to  be  sterilized  is  not  too 
hard  to  filter,  a  common  bottle  aspirator  (Fig-.  7,  a)  can  be 
employed.  The  details  of  the  process  are  then  as  follows: 
The  open  end  of  the  filter  is  closed  by  a  plug-  of  cotton  batting-. 
It  is  then  wrapped  in  paper  and  sterilized  at  150°  C.  The  open 
ends  of  both  tubes  of  the  wash-bottle  are  plugged  with  cot- 
ton, wrapped  in  paper  and  sterilized  with  the  filter,  as  is  the 
plugged  wash-bottle.  The  rubber  stopper  of  the  latter  (bored 


FIG.  7.— Chamberland  Filter  set  up  for  use,  with  Aspirator  and  Wash-bottle  Reservoir. 

with  two  holes  for  the  tubes),  and  the  two  pieces  of  firm-walled 
rubber  tubing-  for  connecting  the  flask  with  the  filter  and  the 
aspirator,  must  be  sterilized  in  some  other  manner.  They  are 
first  laid  for  fifteen  minutes  in  a  -^  per  cent  sublimate  solu- 
tion, rinsed  in  sterilized  distilled  water,  plug-g-ed  with  cotton 
that  has  been  previously  sterilized  by  dry  heat,  and  after 
wrapping-  in  paper  they  are  steamed  for  fifteen  minutes.  After 
this  has  been  done,  the  filter  is  put  together  as  rapidly  as  pos- 
sible, and  with  the  ulmost  cleanliness,  the  fingers  being  first 
washed  in  sublimate.  The  glass  tubes  are  fixed  in  the  rubber 
stopper,  and  this  is  fitted  to  the  wash-bottle  after  removing 
the  plug  from  the  latter  with  sterilized  forceps.  The  long 


Bacteriological    Technology.  1 1 

tube  is  then  joined  to  the  filter  by  one  piece  of  rubber  tubing- 
after  removal  of  its  cotton  plug1.  The  only  plug-  left  in  place 
is  that  at  *  of  Figure  7.  The  entire  apparatus  is  now  steamed 
for  ten  minutes,  after  which  it  can  be  used.  For  additional 
safety,  it  is  usually  customary  after  the  last  sterilization  to 
bind  the  rubber  tubing1  fast  to  the  filter  and  glass  tubes  by 
cord,  or  better  still  by  previously  glowed  coarse  brass  wire, 
which  is  tightly  wound  by  the  use  of  pliers. 

If  instead  of  the  common  wash-bottle,  a  so-called  Pasteur 
flask,  made  entirely  of  glass,  is  used  (cf.  Chapter  XL),  the  pre- 
parations for  filtering1  are  very  much  simplified.  It  is  also 
evident  that  if  an  autoclave  is  at  hand  the  preparatory  steril- 
ization, as  well  as  the  rapid  and  careful  setting-  up  of  the 
apparatus  can  be  dispensed  with.  Without  any  especial  pre- 
cautions the  glass  and  rubber  parts  can  be  joined  and  then 
sterilized  in  a  few  moments  at  120°  C. 

Figure  7  shows  the  apparatus  in  use.  The  filter  is  im- 
mersed in  a  relatively  narrow  glass  cylinder,  which  contains 
the  fluid  to  be  filtered,  as  this  sinks  in  the  glass  it  is  tipped  so 
as  to  use  all  of  the  filtering-  surface  and  to  prevent  air  from 
being  sucked  through,  though  this  is  also  sterilized  in  the  pas- 
sage through  the  filter.  The  simple  aspirator  (a)  is  sufficient, 
when  one  has  not  to  deal  with  fluids  that  are  hard  to  filter. 
The  bubbling-  of  air  back  through  the  aspirator,  and  the  con- 
sequent hindrance  to  filtration,  is  avoided  by  reg-ulating-  the 
size  of  the  escape  tube  by  a  pineh-cock.  In  other  cases,  or 
when  the  filtering  must  be  done  rapidly,  more  powerful  suction 
is  employed,  e.g.,  the  small  air  pump  recommended  by  Cham- 
berland  (Fig.  8,  a),  or  a  filter-pump  worked  by  water.  If  the 
filtration  is  difficult  and  a  powerful  pump  is  used,  care  must 
be  taken  that  the  walls  of  the  flask  are  strong  enough  to  bear 
the  atmospheric  pressure.  In  such  cases  it  is  customary  to 
use  a  thick- walled  flask  instead  of  a  common  wash-bottle,  and 
under  these  circumstances  exceptional  use  may  be  made  of  a 
lead  tube  within  the  rubber  tubing.  After  repeated  sterilizing- 
by  steam,  even  thick-walled  rubber  tubing-  becomes  too  much 
softened  for  further  use. 

When  the  wash-bottle  (b)  is  filled  with  the  clear  filtrate,  it 
is  first  loosened  from  the  aspirator,  then  the  tube  (c)  is  fast- 
ened by  a  clamp  or  glass  plug,  after  which  the  flask  can  be 
separated  from  the  filter  and  its  contents  kept  sterile.  The 


12  Bacteriological    Technology. 

filtrate  remaining-  in  the  cavity  of  the  filter  can  be  saved,  by 
pouring1  it  into  one  or  more  sterile  vessels,  e.g.,  culture  tubes, 
as  soon  as  the  filter  is  opened.  This  is  best  done  by  using  a 
pipette  (Fig.  G,  B),  plugged  at  the  top  with  cotton,  while  the 
tube  below  the  bulb  is  long  enough  to  reach  the  bottom  of  the 
filter  and  slender  enough  to  pass  through  its  opening.  Before 
use,  it  is  hermetically  sealed,  sterilized,  and  passed  through  the 
flame  like  an  ordinary  Pasteur  pipette,  of  which,  indeed,  it  is 
only  an  enlarged  edition  (c/.  Fig.  22). 

After  being  used,  the  Chamberland  filter  is  cleaned  accord- 
ing to  circumstances,  by  simply  brushing  off  the  surface  and 
washing-  it  in  water,  or  by  the  additional  use  of  chemicals, 
e.g.,  disinfectants.  Occasionally  it  may  also  be  desirable  or 
necessary  to  rinse  its  pores  out  thoroughly  with  water,  before 
it  is  dried  and  again  sterilized.  This  is  most  quickly  accom- 
plished (Fig.  6,  C),  in  the  following  way:  the  filter  is  filled 
with  water  and  immersed  in  a  large  vessel  of  water,  which  is 
placed  quite  high,  as  on  a  shelf  or  cupboard.  A  rubber  tube 
several  feet  long  is  attached  to  its  open  end.  Somewhere  in 
this  a  glass  tube  (Fig.  6,  D),  filled  with  water  is  inserted. 
Before  putting  together,  the  filter  and  tube  arc  both  filled 
with  water. 

When  one  has  to  do  with  small  quantities  of  fluid  the  small 
thick- walled  filter  (Fig.  6,  E]  can  be  used.  This  is  15  cm. 
long,  with  a  cavity  2  mm.  in  diameter.  These  were  first  used 
in  Pasteur's  laboratory,  inserted  in  a  filtering  apparatus  of 
special  construction,  but  they  can  be  used  in  the  same  manner 
as  the  larger  ones,  by  fastening  a  rubber  tube  to  the  upper 
end  with  copper  wire.  A  strong  suction  apparatus  is  needed 
for  this.  The  small  bulb  (Fig.  6,  F)  is  intended  to  be  used  as 
a  receiver  of  filtered  fluid  (Fig.  8)  and  also  without  special 
cnange,  as  a  culture  vessel,  with  tubular  plug  (cf.  Chapter 
IL,  p.  16). 

It  must  be  remembered  that  even  filtration  through  porce- 
lain does  not  always  avoid  the  chemical  modification  of  fluids, 
since  the  filter  may  hold  back  certain  compounds,  as  well  as 
the  gerrns.  If  organic  culture  fluids  must  be  absolutely  un- 
changed, and  at  the  same  time  free  from  g-erms,  it  is  neces- 
sary to  do  without  all  sterilization,  in  which  case  they  are  to 
be  collected  or  prepared  with  such  care  that  all  contamina- 
tions from  their  surroundings  are  avoided.  Blood  and  urine. 


Bacteriological    Technology.  1 3 

for  instance,  are  drawn  directly  from  the  heart,  veins,  or  blad- 
der with  antiseptic  precautions.  The  preparation  of  origin- 
ally sterile  infusions  is  further  briefly  discussed  in  Chapter  XI. 
5.  Disinfecting  Solutions. — As  a  help  in  sterilization,  it  is 
usual  to  have  at  hand  a  large  quantity  of  corrosive  sublimate 
solution  (1  or  2 : 1,000),  which  is  not  only  used  in  preparing  some 
sorts  of  culture  media,  e.g.,. potatoes  (see  Chapter  III.),  but  in 
many  other  cases,  for  cleansing  the  hands,  instruments,  mor- 
bid material,  etc.  If  well  water  is  used  instead  of  distilled 
water  in  making  this  solution,  care  must  be  taken  that  no 
precipitate  of  insoluble  mercurial  salts  is  formed,  or  the  fluid 


FIG.  8.— Small  Porcelain  Filter  joined  to  Bulb-reservoir  and  Air-pump  (a). 

will  possess  no  disinfecting  value.  To  prevent  this,  a  small 
quantity  of  acetic  acid  may  be  added  to  the  well  water.  Fiir- 
bringer  recommends  0.5  gram  to  each  litre  of  0.1  per  cent  solu- 
tion. Carbolic  acid  solutions  of  various  strengths  are  also  to 
be  kept  ready  for  use. 


CHAPTER  II. 

COMMON  CULTURE-APPARATUS. 

THE  principal  requirements  of  a  culture-apparatus  in  which 
pure  cultures  are  to  be  conveniently  and  surely  obtained  are 
the  following1:  Besides  being-  durable,  simple,  handy,  and 
cheap,  it  must  be  easily  sterilized  with  its  contents,  and  so 
that  it  can  be  opened  for  inoculation  and  again  closed  without 
much  danger  of  infection  from  the  air.  It  must  also  be  so 
closed  that  the  entrance  of  foreign  germs  is  impossible,  while 
free  access  of  the  air  is  permitted  (except  when  anaerobic 
forms  are  to  be  cultivated).  This  is  effected  by  the  use  of  cot- 
ton batting-,  which  allows  a  sufficient  circulation  of  air  while 
filtering  the  germs  from  this. 

The  apparatus  to  be  recommended  for  simply  keeping  a 
pure  culture  is  the  following- : 

1.  Test-tubes  and  flasks  plugged  with  cotton.  One  can 
commonly  use : 

a.  Test-tubes  of  any  size.  The  use  of  small  tubes  (e.g.,  13 
cm.  long,  and  1  cm.  in  diameter)  is  to  be  recommended.  A 
considerable  quantity  of  the  culture  material  is  then  saved. 
Since  the  heating-  of  these  glasses  is  generally  effected  in  the 
steam  sterilizer  and  not  directly  over  the  flame,  their  walls 
need  not  necessarily  be  so  thin  as  those  of  chemical  test-tubes, 
and  consequently  it  may  be  best  to  order  those  with  especially 
thick  walls,  entirely  without  a  flange,  so  that  the  cotton  plug- 
may  fit  better  to  the  edge. 

The  test-tubes  are  first  carefully  cleaned  with  common 
distilled  water,  and  then  plugg-ed  in  the  following  manner: 
Good  cotton  batting  (not  absorbent  cotton)  is  pressed  with  a 
pair  of  forceps  (or  twisted  by  the  hand)  into  the  mouth  of  the 
test-tube  in  sufficient  quantity  to  form  a  firm,  tightly  fitting 
plug,  which  reaches  a  couple  of  centimetres  into  the  tube, 
while  part  of  it  projects  and  frays  out  over  the  edge.  The 


Bacteriological    Technology. 


5 


plug-  must  be  both  firm  and  tightly  fitting,  yet  capable  of  re- 
moval and  reinsertion  (by  a  twisting  motion)  without  much 
difficulty.  When  the  test-tubes  have  been  plugged,  they  are 
put  in  a  small  four-sided  basket,  made  of  wire-cloth,  of  a  suita- 
ble size  for  the  oven,  and  sterilized  at  150°  C. 

If  it  is  wished  to  make  use  of  a  larger  surface  than  is  avail- 
able when  a  test-tube  is  used,  even  when  it  is  obliquely  placed 
(Fig.  9,  II.),  recourse  is  had  to : 

b.  Small  Conical  Flasks. — (Erlenmeyer  flasks)  of  the  form 
shown  in  Fig.  9,  VII.,  and  holding  about  100  cc. 


FIG.  9. -Various  Culture-glasses  with  Cotton  Plugs  (/.,  IL,  VII.),  or  Plugged  Caps  (III.— FI.) 

c.  Small  Medicine  Bottles  (30  gm.)  are  conveniently  used 
in  the  same  way,  and.  the  surface  of  the  gelatin  in  them  can 
also  be  increased  by  placing  them  in  an  oblique  position  while 
it  is  hardening.  They  have  the  advantage  over  test-tubes 
that  they  may  be  stood  upright  without  the  use  of  a  rack. 

Any  of  these  receptacles  can  be  used  for  cultures  in  either 
solid  or  fluid  media.  A  fault  common  to  them  is  that  they 
are  all  relatively  wide  mouthed,  so  that  the  removal  of  the 
plug  leads  to  a  certain  danger  of  contamination,  especially 
from  dust  which  has  settled  on  the  plugs  when  they  have 
stood  for  some  time.  This  can  be  obviated  by  tying  a  couple 


1 6  Bacteriological    Technology. 

of  layers  of  filter  paper  over  the  cotton  plug1  (Fig.  9,  VIL), 
which  is  especially  recommended  when  large  flasks  are  used. 
For  greater  safety,  the  part  of  the  cotton  projecting  beyond 
the  tube  can  be  singed  off  just  before  use.  Both  these  precau- 
tions are  less  necessary  when  solid  media  are  used,  so  that  the 
tube  or  flask  can  be  inverted  while  the  plug  is  removed  and 
inoculation  effected.  They  are  more  important  when  the  con- 
tents of  the  vessels  are  fluid.  Much  greater  safety  is  secured 
by  the  tubular  cotton  plug  of  the  author  which  is  described 
later. 

2.  Vessels  with  Tubular  Plugs. — When  I  began  in  1879-80 
the  pure  culture  of  a  large  number  of  putrefactive  bacteria, 
which  had  been  isolated  by  the  capillary-tube  method,  I  first 
used  stoppers  in  the  form  of  short  pieces  of  rubber  tubing,  in 
one  end  of  which  small  sterilized  tampons  of  cotton  were  fixed. 
The  vessels  which  were  closed  by  these  tubular  stoppers  were 
larger  and  smaller  flask-shaped  or  test-tube-shaped  glasses, 
which  had  a  short  and  broad  slightly  conical  neck,  with  rela- 
tively narrow  mouth  (2  to  4  mm.)  over  which  the  rubber  tube 
could  be  slipped  (Fig.  9,  III.-V.). 

The  tubular  stoppers  are  prepared  as  follows :  The  cotton  is 
sterilized  at  150°  C.  The  rubber  tube,  6  to  8  cm.  long,  is  steril- 
ized by  steam,  wrapped  in  paper.  It  should  be  a  little  larger 
than  the  top  and  a  little  smaller  than  the  bottom  of  the  conical 
neck  it  is  to  fit.  After  cooling,  the  tubes  are  unwrapped,  and 
by  the  aid  of  small  forceps,  half  filled  with  tampons  of  the 
sterilized  cotton,  which  must  be  so  large  and  firm  that  they 
bulge  the  tube  slightly  (Fig.  9,  V.).  The  advantages  of  these 
stoppers  are,  obviously :  a.  In  opening  and  closing  the  flasks 
the  cotton  and  the  dust  that  has  collected  on  it  are  not 
touched.  6.  The  apparatus  is  opened  and  closed  at  a  point 
where  its  surface  can  always  be  easily,  freed  from  dust.  c. 
The  opening  through  which  inoculation  is  effected  is  smaller 
than  with  test-tubes. 

The  same  principle  was  afterward  applied  in  a  more  ade- 
quate form  in  the  so-called  Pasteur  or  Chamberland  flasks 
(Fig.  10)  which  are  much  used  in  Pasteur's  laboratory.  The 
cap  of  these  is  not  made  of  rubber  but  of  glass,  and  is  closely 
fitted  to  the  neck  by  grinding.  Unlike  the  flasks  with  rubber 
tubes,  they  can  be  sterilized  entire  at  150°  C.,  and  hence,  as  a 
rule,  are  preferable  to  the  latter.  The  flasks  and  caps,  which 


Bacteriological   Technology.  17 

belong  together,  are  numbered  correspondingly  with  Bruns- 
wick black  (Fig.  10). 

Notwithstanding  the  decided  superiority  of  the  tubular  cap 
over  the  simple  cotton  plug,  the  latter  as  a  rule  is  used  even 
in  fluid  cultures;  but  the  rubber  cap  is  always  useful  when  a 
narrow  tube  is  to  be  plugged  with  cotton  (cf.  Fig.  56  and  Fig. 
58).  By  means  of  a  bored  rubber  stopper,  a  tubular  cap  can 
be  adapted  to  any  flask  (Fig.  9,  VI.). 

3.  Other  Culture  Apparatus. — A  pair  of  small  glass  trays 
(3  to  5  cm.  in  diameter)  can  be  used  for  cultures,  one  serving  as 
a  lid  for  the  other  (Fig.  13).  They  have  the  advantage  over 


FIG.  10.— Pasteur-Chamberland  Flask. 


Fio.  11.— Glass  Box  for  Soyka's 
Museum  Cultures. 


vessels  plugged  with  cotton,  that  their  contents  are  more 
easily  accessible,  even  for  microscopic  examination,  but  they 
are  less  secure  against  foreign  germs. 

A  far  more  reliable  security  against  contamination  is 
afforded  by  glass  boxes  with  overlapping  and  ground  covers 
(Fig.  11),  such  as  Soyka  uses  for  his  hermetically  sealed  "mu- 
seum cultures"  (cf.  Chapter  VI.). 

A  number  of  .other  contrivances  for  cultures  adapted  to 
particular  purposes  will  be  described  later.  The  apparatus 
for  cultivating  anaerobic  bacteria,  and  for  cultivating  bacteria 
under  the  microscope,  are  for  convenience  left  for  treatment 
in  later  chapters,  devoted  to  these  subjects. 


CHAPTEE  III. 

CULTURE-MEDIA,  AND  THEIR  INTRODUCTION  INTO  TEST- 
TUBES,   FLASKS,   ETC. 

IT  would  be  useless  to  mention  the  enormous  number  of 
solutions,  infusions,  etc.,  which  have  been  applied  advantage- 
ously in  various  bacteriological  investigations,  and  moreover 
it  may  obviously  be  necessary  for  each  experimenter  to  use 
the  most  dissimilar  culture-media,  and  to  vary  them,  within 
wide  limits,  by  the  addition  or  removal  of  one  substance  or 
another  according-  to  the  object  of  the  investigation.  Direc- 
tions are  given  here  for  the  preparation  of  only  such  fluid  and 
solid  media  as  have  been  found  especially  useful  in  the  study 
of  pathogenic  micro-organisms  during  the  last  few  years. 
They  are  commonly  made  in  the  conical  Erlenmeyer  flasks 
(Fig.  10,  VII.).  These  conical  flasks  will  be  frequently  men- 
tioned in  the  following  pages,  where  they  are  designated  as 
large,  medium,  and  small,  holding  respectively  1,500,  500,  or 
100  gin. 

A.  FLUID  MEDIA. 

1.  Flesh  Water. — Even  the  simple  decoction,  made  by  cook- 
ing meat  in  water,  with  an  acid  reaction,  affords  a  good  nutri- 
ent substance  for  a  large  number  of  bacterian  forms,  but 
when  it  is  neutralized  by  the  addition  of  a  little  sodium  carbon- 
ate, a  fluid  is  obtained  in  which  a  large  number  of  the  most 
important  pathogenic  germs  yet  known,  also  thrive.  For  the 
preparation  of  broth,  different  kinds  of  meat  are  used  accord- 
ing to  circumstances,  and  the  procedure  is  somewhat  different 
in  the  various  laboratories.  This  is  worth  noting,  because 
we  must  be  prepared  to  recognize  in  our  present  imperfect 
knowledge  concerning  the  nutrition  of  bacteria,  that  slight 
variations  in  the  preparation  may  give  rise  to  differences  in 
the  results  of  cultures. 


Bacteriological    Technology.  19 

The  two  most  common  methods  of  preparation  are  the 
following- : 

a.  Bouillon  (B). —  A  pound   of  lean,   scraped,   or  finely 
chopped  beef,  is  placed  in  an  enamelled  kettle,  or  a  large  flask, 
with  a  litre  of  distilled  water.     The  mixture  is  cooked  for  half 
an  hour  and  filtered.     The  filtrate  is  neutralized  or  rendered 
slightly  alkaline  by  adding-  to  it,  drop  by  drop,  a  solution  of 
carbonate  (or  phosphate)  of  sodium.     It  is  then  again  boiled 
for  about  an  hour,  by  which  time  the  insoluble  albuminoids 
are  all  coagulated  (C.  Fraenkel),  after  which  it  is  allowed  to 
cool,  when  the  fat  solidifies.     Having  been  once  more  filtered, 
the  clear  broth  is  filled  into  small  vessels  (usually  medicine 
bottles   [or  test-tubes],  plugged  with  cotton,   c/.   p.  21),    in 
which  it  is  finally  sterilized  in  the  steam-cylinder  for  a  quarter 
of  an  hour.     For  greater  security,  it  is  again  sterilized  on  the 
following  day   (disconnected   sterilization,  c/.    p.  7).      With 
each  new  cooking,  the  clear  filtered  broth  may  become  clouded, 
but  the  turbidity  disappears  on  cooling,  otherwise  it  is  neces- 
sary to  repeat  the  cooking  and  filtration  several  times,  until 
the  fluid  is  obtained  perfectly  clear.     This  decoction  of  meat 
is  known  in  the  laboratory  as  bouillon  (B)  in  contrast  with  the 
following. 

b.  Flesh  water  (K),  in  the  more  restricted  sense.     A  pound 
of  chopped  lean  beef  is  covered  with  a  litre  of  water  and  set 
in  an  ice-chest  for  twenty-four  hours,  after  which  it  is  thor- 
oughly shaken  and  filtered  through  muslin,  the  juices  being 
well  wrung  out  from  the  meat.     In  this  way  a  litre  of  flesh 
extract  is  obtained,  which  is  then  cooked,  filtered,  rendered 
slightly  alkaline,  etc.,  as  before. 

The  addition  of  0.5  per  cent  of  table  salt  increases  the  value 
of  broth  and  flesh  extract  as  a  culture  fluid  for  a  number  of 
bacteria  (Miquel). 

The  addition  of  five  per  cent  of  glycerin  (before  the  last 
neutralization)  gives  an  excellent  medium  for  the  tubercle 
bacillus  (Roux  and  Nocard). 

Obviously,  there  may  also  be  occasion  for  varying  the  com- 
position of  the  bouillon  by  the  addition  of  various  other  sub- 
stances, e.g.,  peptone,  cane  or  grape-sugar,  acetic  acid,  etc. 

A  useful  flesh-water  can  also  be  obtained  by  the  solution  in 
water  of  a  suitable  quantity  of  some  meat-extracts,  followed 
by  very  careful  "  disconnected "  sterilization  (because  of  the 


2O  Bacteriological    Technology. 

numerous  resistant  germs  often  present  in  these  extracts), 
neutralization,  filtration,  etc. 

c.  Liebig's  extract  (E),  5  gm.  to  a  litre  of  water,  is  to  be 
recommended,  and  especially 

d.  Cibil's  extract  (C),  20  gm.  to  a  litre  of  water 

Of  other  nutrient  fluids,  only  the  following-,  of  proved  and 
recognized  value,  will  be  named  : 

2.  Aqueous  decoction  of  liver,  lungs,  and  other  viscera. 

3.  Neutralized  or  slightly  alkaline  wine  (used  at  one  time 
by  Pasteur)  in  the  pure  cultivation  of  the  bacillus  of  splenic 
fever,  on  a  large  scale. 

4.  Infusion  or  decoction  of  wheat,  hay,  cabbage. 

5.  Yeast- water,  a  filtered  and  sterilized  decoction  of  100 
parts  water  to  10  parts  compressed  yeast. 

For  the  cultivation  of  yeast  and  moulds,  the  following  are 
especially  adapted : 

6.  Beer-wort,  a  decoction  of  dried  and  pulverized  malt,  ob- 
tainable at  every  brewery.     This  must  be  cooked  for  an  hour 
and  then  filtered,  before  use,  but  it  is  hard  to  obtain  it  clear. 

7.  Decoction  of  horse  dung  (and  of  the  excrement  of  other 
herbivora).     One  part  of  fresh  horse  dung  is  mixed,  by  the  use 
of  a  glass  rod,  with  three  parts  of  water,  set  in  a  cool  place  for 
twenty-four  hours,  after  which  the  mixture  is  cooked  for  an 
hour,  and  filtered  through  a  double  filter,  which  is  a  very  slow 
process  (unless  a  filter  pump  is  used).     The  filtrate  is  again 
cooked  for  some  time  and  if  necessary  refiltered,  after  which 
it  is  filled  into  small  receptacles,  sterilized    in  the   steam- 
cylinder,  and  boiled  twice  for  fifteen  minutes,  with  a  day  be- 
tween. 

8.  Decoction  of  prunes,  which  is  best  prepared  as  follows  : 
The  prunes  are  allowed  to  stand  for  a  day  in  a  little  water,  in 
wrhich  they  are  then  cooked  carefully,  so  that  they  remain 
unbroken.     The  fluid  is  afterward  filtered   and  boiled  down 
somewhat.     In  some  cases  it  may  be  desirable  to  reduce  the 
acidity  of  the  decoction  by  the  addition  of  sodium  phosphate. 

9.  Decoction  of  other  dried  fruits,  e.  g.,  raisins,  dried  pears, 
etc. 

The  horse  dung,  prunes,  raisins,  etc.,  used  in  making  these 
decoctions,  may  be  preserved  in  a  sterile  condition,  for  use  as 
solid  media  in  the  culture  of  moulds,  yeasts,  etc. 

A  neutral   or  very  slightly  acid  beer-wort  is  especially 


Bacteriological    Technology.  2 1 

adapted  to  the  culture  of  Mucorini;  while  for  the  various 
species  of  Aspergillus  a  simple  acid  mixture  of  wort  and 
prune-juice  is  an  especial!}^  good  medium,  as  O.  Joh.  Olson 
has  told  me.  A,  mixture  of  the  three  liquids  numbered  6,  7, 
and  8,  may  at  times  be  useful.  The  reader  is  referred  to 
Chapter  XL  for  directions  for  collecting-  and  preparing  prima- 
rily sterile  nutrient  fluids. 

When  a  large  quantity  of  culture  fluid  has  been  prepared, 
it  is  distributed  in  several  medicine  bottles  (or  Erlenmeyer 
flasks)  holding  one  or  two  hundred  grams,  previously  plugged 
with  cotton  and  sterilized  at  150°  C.  In  these  smaller  vessels 
the  fluid  is  finally  sterilized  in  the  steam  cylinder  for  five  to 
fifteen  minutes  on  two  sucessive  days.  It  can  then  be  pre- 
served as  long  as  is  wished,  in  a  dry  place,  provided  the  flasks 
are  well  plugged.  The  safest  plan  is  to  use  a  large  cotton 
plug,  and  to  tie  over  it  several  layers  of  filter  paper  (or  to 
cover  it  with  a  sterilized  thin  rubber  cap,  such  as  the  Germans 
now  use  extensively). 

Formerly  "Pasteur's  fluid"  (pure  rock-candy,  10  gm;  am- 
monium acetate,  0.1  gm.;  and  the  ashes  of  1  gm.  yeast,  all  dis- 
solved in  distilled  water),  "  Mayer's  fluid,"  or  "  Cohn's  fluid  " 
(potassium  phosphate,  0.5  gm.;  magnesium  sulphate,  0.5  gm.; 
tribasic  phosphate  of  potassium,  0.5  gm. ;  acetate  of  ammonium, 
1  gm. ;  water,  100  gm.)  were  used.  These  fluids  are  little  suited 
to  the  cultivation  of  bacteria.  It  must  also  be  remembered 
that  Pasteur  by  no  means  introduced  such  mineral  solutions  as 
suitable  nutrient  fluids  for  microbes,  but  to  show  (1858)  that 
yeast  cells  can  produce  albuminoids  from  a  carbo-hydrate  and 
an  inorganic  nitrogenous  compound,  when  the  necessary  ash- 
constituents  are  also  present. 

B.  SOLID  MEDIA. 

The  systematic  use  of  solid  nutrient  media,  especially  of 
nutrient  gelatin  (Robert  Koch,  1881),  marks  a  turning  point  in 
the  history  of  bacteriological  technology.  Brefeld  had  previ- 
ously employed  nutrient  gelatin,  but  essentially  only  to  check 
the  drying  of  slide-cultures.  The  starting  point  for.  Koch's  im- 
portant discovery  was  the  long-known  fact  that  the  cut  surface 
of  a  cooked  potato,  laid  away  for  some  time  exposed  to  the 
air,  becomes  the  seat  of  large  and  small  colonies  of  mould, 


22  Bacteriological    Technology. 

yeast,  and  bacteria,  the  last  two  of  which  often  occur  as 
small  distinct,  slimy  colonies  of  various  colors.  Each  colon}^ 
as  a  rule,  contains  only  one  form  of  yeast  or  bacteria,  which 
has  developed  from  a  germ  that  fell  from  the  air  and  found 
in  the  potato  a  favorable  soil  for  its  growth  (first  noticed  by 
Hoffmann,  in  1869,  and  utilized  by  Schroeter  in  1872,  in  his 
cultivation  of  pigment  bacteria).  If  we  imagine  these  germs 
to  have  fallen,  not  on  the  solid  surface  of  the  potato,  but  on 
an  equally  large  surface  of  some  fluid  in  which  they  could 
thrive  as  well,  it  is  easily  seen  that  the  several  forms  would 
have  run  together  after  a  short  time,  motile  and  quiescent 
being  mixed  together.  Some  of  the  germs  which  succeeded 
in  developing  upon  the  potato,  where  they  found  space  un- 
disturbed by  other  colonies,  would,  perhaps,  have  failed  to 
develop  at  all  in  the  fluid,  yielding  to  others  in  the  struggle 
for  existence.  The  same  germs  which  in  the  fluid  produced 
a  motley  tangle  of  intermingled  forms,  gave  a  series  of  well- 
separated  colonies,  on  the  solid  medium. 

When  Koch  had  become  aware  of  the  extraordinary  ad- 
vantage offered  by  cultures  on  solid  media  over  those  in  fluids, 
he  sought  to  give  various  useful  culture  fluids  a  solid  form, 
and  he  succeeded  in  doing  this  by  gelatinizing  them,  in  the 
manner  to  be  described.  The  "nutrient  gelatins"  so  pre- 
pared, have  the  advantage  over  potato,  that  their  chemical 
composition  can  be  varied  within  wide  limits,  so  that  solid 
culture  media  may  be  produced  for  such  bacteria  as  cannot  be 
found  on  potato.  They  are,  further,  transparent,  which  ren- 
ders possible  the  observation  of  the  growth  of  bacteria  within 
the  gelatin,  as  well  as  the  microscopic  examination  of  the 
culture.  Finally,  they  are  liquefiable  at  a  low  temperature, 
which  is  of  decided  value  for  their  application  to  the  isolation 
of  the  different  bacterian  germs. 

A  low  melting  point,  however,  limits  in  some  ways  the  use- 
fulness of  gelatins,  since  it  makes  it  impossible  to  employ 
them  for  cultures  at  much  above  20°  C.  Koch  therefore  in- 
troduced for  such  cultures  a  second  gelatinizing  substance, 
agar-agar,  which  remains  solid  at  the  highest  temperatures 
used  for  culture  investigations.  The  same  remark  applies  to 
sterilized  blood-serum,  which  Koch  found  a  method  of  pre- 
paring. 

It  was  said  above  that  the  introduction  of  solid  culture- 


Bacteriological    Technology.  23 

media  marks  a  turning-  point  in  the  history  of  bacteriological 
technology.  This  is  doubly  true.  In  the  first  place,  it  has  be- 
come possible  by  them  to  surely  and  easily  isolate,  and  culti- 
vate in  a  state  of  purity,  the  various  bacterian  forms,  as  is 
evident  from  what  has  been  said,  and  as  will  be  shown  more 
in  detail  in  the  next  chapter.  But  the  introduction  of  solid 
media  has  also  in  many  ways  simplified  bacteriological  work, 
making  it  possible  to  work  with  far  simpler  apparatus,  and  at 
the  same  time  with  far  better  control  and  far  greater  cer- 
tainty, aside  from  other  reasons,  because  any  accidentally  in- 
troduced foreign  germ  manifests  itself  more  readily  when  it 
forms  a  limited  colony  on  or  in  the  gelatin,  than  when  its 
progeny  in  a  fluid  become  scattered  and  mixed  among-  all  the 
other  bacteria. 

It  must  not  be  forgotten,  however,  that  there  are  also  limits 
to  cultivation  upon  solid  media,  and  that  it  by  no  means  neces- 
sarily renders  fluid  cultures  superfluous;  so  that  the  same 
care  is  due  to  the  technique  of  the  latter  as  formerly.  To  men- 
tion a  single  instance  of  many :  it  is  obvious  that  experiments 
concerning  the  fermentation  products  of  bacteria,  and  their 
nutrition,  may  demand  the  use  of  culture  fluids  which  have  a 
very  simple  chemical  composition,  but  which  would  be  changed 
into  very  troublesome  and  complex  mixtures  by  the  addition 
of  gelatin  or  agar-agar.  In  addition  to  this,  the  usefulness 
of  a  culture  fluid  for  certain  bacteria  is  at  times  lessened  or 
destroyed  by  the  addition  of  gelatin,  etc. 

1.  Boiled  Potato. — According-  to  circumstances,  the  pre- 
paration and  application  of  potatoes  varies  a  little,  so  that  one 
either : 

1.  Simply  cuts  them  in  two,  and  lays  them  in  a  moist 
chamber.  The  problem  in  preparing-  them  is  first  and  chiefly, 
to  remove  or  sterilize  the  dirt  adhering  to  them,  which  always 
contains  larg-e  numbers  of  particularly  resistant  bacillus  spores. 
To  this  end  as  clean  and  smooth-skinned  potatoes  as  possible 
are  selected.  Then  they  are  repeatedly  washed  in  water,  and 
any  remaining-  dirt  is  carefully  scrubbed  off  under  water,  with 
a  brush.  By  the  use  of  a  pointed  knife  any  diseased  or  injured 
pa^ts  of  the  skin,  as  well  as  the  changed  parts  of  the  under- 
lying- parenchyma,  are  removed.  The  potatoes  are  then  laid 
for  some  time  in  a  0.1  per  cent,  sublimate  solution,  wrapped 
singly,  without  being-  dried,  in  thin  wrapping  paper,  and  ex- 


24  Bacteriological    Tcclinology. 

posed  for  half  an  hour  to  streaming-  steam  at  100°  C.  After 
twent3^-four  hours  the  steaming-  is  repeated  for  fifteen  minutes 
and  the  potatoes  are  ready  for  use.  They  are  taken  from  the 
paper  one  at  a  time,  held  between  thumb  and  one  finger  of 
the  left  hand,  which  has  previously  been  washed  in  sublimate, 
and  are  halved  by  a  table  knife  that  has  been  carefully  steril- 
ized by  being  drawn  several  times  through  the  flame,  or  by 
prolong-ed  heating-  at  150°  C.  (wrapped  in  paper  in  the  steril- 
izing- oven).  The  halves  are  then  quickly  laid  with  the  cut 
faces  up,  in  a  moist  chamber,  e.g.,  under  a  bell  glass  set  on  an 
earthen  plate  (or  in  the  flat  pairs  of  trays  similar  to  Fig-.  13, 
but  about  20  cm.  in  diameter  which,  though  they  cannot  be 
so  easily  obtained,  are  safer  and  have  the  advantage  that 
they  can  be  set  away  one  on  top  of  the  other).  Dish  and  bell 
glass  have  first  been  carefully  cleaned  in  water  and  rinsed  in 
1.0  per  cent  sublimate;  and  one  or  two  thicknesses  of  filter 


FIG.  12.  FIG.  13. 

FIGS.  12  and  13.— Shallow  and  Deep  Glass  Trays  in  Pairs,  the  Larger  serving  as  a  Lid  for 

the  Smaller. 

paper  moistened  in  the  same  solution  have  been  laid  in  the 
bottom  of  the  plate.  A  potato  cooked  and  divided  in  this 
manner,  placed  under  a  bell  glass  is  one  of  the  simplest  ar- 
rangements for  cultivating  bacteria,  but  it  is  always  exposed 
to  contamination  by  germs  from  the  air.  This  is  avoided  by: 

2.  Dividing  the  potatoes  into  prismatic  pieces  (or  cylinders, 
by  the  use  of  a  small-sized  tin  cutter  made  like  common  apple 
corers)  and  putting  these  in  plugged  test-tubes.     In  this  case 
it  is  only  necessary  to  cleanse  the  surface  of  the  potato  with 
brush  and  sublimate  solution.    After  the  potatoes  have  been 
steamed  once  for  five  minutes,  they  are  pared  and  with  sterile 
instruments  cut  into  pieces  which  are  quickly  placed  in  the 
test-tubes  already  sterilized  in  the  usual  way,  after  which  they 
are  steamed  for  a  quarter  of  an  hour. 

3.  Nicer  cultures  are  obtained  by  cutting  the  cooked  and 
pared  potato  into  round  discs,  which  are  laid  in  the  bottom 
of  a  small  pair  of  glass  trays  (Fig.  13,  cf.  Fig.  11,  as  well  as 
Soyka's  museum  cultures),  which  after  being  wrapped  in  paper 


Bacteriological    TecJmology.  2  5 

are  then  steamed  for  fifteen  minutes.  Such  discs  are  best  cut 
out  by  aid  of  a  small  tin  ring1. 

4.  Potato  broth  prepared  by  mashing-  pared  boiled  pota- 
toes and  adding-  a  proper  amount  of  water  can  be  occasionally 
used  with  advantage,  if  an  especially  large  culture  surface  is 
desired. 

2.  Gelatinized  Media. — If  we  desire,  according  to  Koch's 
directions,  to  change  our  different  culture  fluids  into  solid  and 
transpsarent  but  liquefiable  substances  we  make  use  of  the- 
f  olio  wing: 

a.  Q-elatin. — The   finest    French  gelatin,  which   comes   in 
thin  oblong  sheets  of  about  2.5  gm.  weight,  is  used.     Five,  or 
commonly  ten,  per  cent  of  this  is  added  to  the  nutrient  fluid, 
dissolved,  cooked,  rendered  slightly  alkaline,  cleared,  filtered, 
poured  into  smaller  vessels  and  steamed  twice  with  a  day's 
interval. 

b.  Agar-agar  (The  Asiatic  name  of  several  peculiar  gela- 
tinous alga2;,  which  grow  in  the  Indian  Archipelago  and  come 
into  the  market,  dried  in  yellowish  cartilaginous  strips  [or 
spongy  prisms] ). — When  cooked  in  water  this  forms  a  stiff 
jelly  and  can  be  added  to  the  various  culture  fluids  in  a  quan- 
tity of  1  to  2  per  cent,  precisely  like  gelatin.     As  a  rule  we  use 
1.5  per  cent,  which  is  dissolved  lay  long  cooking  and  rendered 
slightly  alkaline,  after  which  it  is  cleared,  filtered,  etc. 

In  passing  to  a  fuller  description  of  the  preparation  of  the  gel- 
atinizing substances,  we  must  dwell  briefly  on  the  advantages 
and  disadvantages  attending  the  use  of  each.  Agar-agar  was 
introduced  b}'  Koch,  as  above  indicated :  (a)  because  it  melts 
at  a  much  higher  temperature  than  gelatin,  and  can  there- 
fore be  used  for  cultures  on  a  solid  medium  at  a  higher  tem- 
perature (30°-40°  C.  or  higher) ;  (b)  it  has  also  the  advantage 
as  compared  with  gelatin  that  it  endures  cooking  for  a  longer 
time  without  diminution  of  its  gelatinizing  power;  (c)  there 
are  many  bacteria  which  liquefy  gelatin  in  their  growth,  but 
do  not  affect  agar  in  this  manner,  which  in  many  cases  is  a 
great  advantage,  especially  in  isolation -cultures.  On  the 
other  hand,  it  must  be  said:  (a)  that  because  of  its  low  melt- 
ing point,  gelatin  is'  better  adapted  to  the  isolation  of  germs ; 
(6)  it  gives  a  filtrate  as  clear  as  water,  while  it  is  very  diffi- 
cult, not  to  say  impossible,  to  get  perfectly  clear  nutrient  agar; 
(c)  the  difference  between  the  mode  of  growth  of  different 


26  Bacteriological    Technology* 

bacteria  shows  far  more  clearly  in  gelatin  than  in  agar,  so 
that  two  species  which  give  in  gelatin  colonies  of  very  differ- 
ent appearance,  sometimes  appear  identical  when  they  have 
grown  in  agar. 

c.  Agar-Grelatin. — To  Jensen  is  due  the  credit  of  combin- 
ing the  good  qualities  of  both  media,  and  avoiding  their  disad- 
vantages, by  adding  to  culture  fluids  5  per  cent  of  gelatin, 
and  0.75  per  cent  of  agar.     The  introduction  of  this  mixture 
marks  a  real  advance,  and  it  is  worthy  of  use  as  almost  the 
chief  culture-medium,  since  it  is  easily  filtered  clear,  and  lique- 
fiable  at  so  low  a  temperature  that  it  can  be  used  without 
difficulty  for  plate-cultures,  though  it  remains  solid  at  30°- 
40°  C. 

d.  Irish-Moss  (Chondrus  crispus),  rarely  used.  —  Neisser 
recommends  a  strength  of  2.5  per  cent. 

The  preparation  of  gelatins,  agars,  and  agar-gelatins,  is 
effected  essentially  in  the  same  manner,  so  that  a  single  de- 
scription will  suffice  for  all.  But  the  length  of  time  during 
which  the  solutions  can  be  kept  at  the  boiling  point  for  steril- 
ization, cleaning,  etc.,  must  be  relatively  short  for  those  con- 
taining gelatin,  which  otherwise  loses  its  powder  of  solidifying. 
Agar,  on  the  other  hand,  endures  long  cooking,  while  for  ag'ar- 
gelatin,  a  golden  mean  is  kept.  Gelatin,  as  a  rule,  is  cooked 
ten  minutes  before  filtration,  and  ten  minutes  after.  Agar  is 
best  cooked  forty-five  minutes  before,  and  a  like  time  after; 
and  agar-gelatin  twenty  minutes  before  and  thirty  minutes 
after  filtering.  Further  than  this,  only  clarifying  and  filter- 
ing demand  special  mention. 

Clarifying. — When  the"  nutrient  jelly  has  cooked  long 
enough,  it  is  allowed  to  cool  to  somewhere  about  50°  C.  An 
egg  is  then  broken  into  100  gm.  water,  and  gradually  added 
to  the  cooled  but  still  fluid  mixture,  with  which  it  is  thor- 
oughly incorporated.  For  a  litre  of  jelly,  the  entire  egg  is 
used,  a  correspondingly  smaller  quantity  being  used  for  less 
than  a  litre.  When  the  mixture  is  again  heated  to  the  boil- 
ing point,  the  white  of  egg  is  precipitated  in  a  large  yellowish 
curd,  floating  in  a  perfectly  clear. fluid.  After  cooking  for 
some  time,  the  next  step  is  proceeded  to — 

Filtering. — This  must  necessarily  be  done  while  the  jelly 
is  still  warm.  If  little  is  to  be  filtered,  it  is  merely  necessary 
to  heat  it  up  well  before  pouring  it  over  the  filter,  so  that,. 


OF  THB 

DIVERSITY 


Bacteriological    Technology. 


27 


notwithstanding-  the  cooling,  it  will  remain  fluid  long-  enough 
for  all  to  run  through.  But  in  this  case,  both  the  funnel  and 
flask  need  to  be  first  warmed.  It  is  very  easy  to  warm  and 
sterilize  flask,  funnel,  and  filter,  as  well  as  to  moisten  the 
latter,  in  the  manner  shown  in  Fig.  14.  A  layer  of  water  one 
or  two  centimetres  deep  is  poured  in  the  flask,  and  the  funnel 
and  filter  are  set  in  its  mouth,  the  top  of  the  funnel  being 
covered  with  several  thicknesses  of  filter  paper,  over  which  a, 
plate  of  zinc  or  asbestos  is  laid  (glass  is  apt  to  break).  By 
heating  the  water  to  the  boiling  point  for  a  few  minutes, 
everything  is  sterilized,  warmed,  and  moistened,  in  a  single 
operation.  While  the  water  is  still  hot, 
it  is  poured  out,  and  filtration  can  begin ; 
the  funnel  being  kept  covered  with  a  zinc 
or  asbestos  plate  which  prevents  cooling 
to  a  considerable  extent. 

When  larger  quantities  (e.g.,  a  litre) 
are  to  be  filtered  at  once,  there  is  danger 
that  the  gelatin  may  stiffen   before  the 
completion  of  the  process,  even  though  it 
was  at  first  almost  boiling.    This  is  pre- 
vented by  using  a  hot- water  funnel  (Fig. 
15)   a  double-walled  water  bath  with  a 
projecting  arm  at  b.    The  apparatus  is 
FIG.  i4.— simple  Filtering    filled  with  water  through  the  hole  a,  and 
Arrangement.  ig  kept  vvarm  throughout  the  filtration  by 

means  of  a  flame  set  under  b.  Such  a  double-walled  funnel 
can  be  made  by  any  tinner,  and  is  far  more  convenient  than 
the  single- walled  Plantamour  funnel  supplied  by  dealers  in 
apparatus,  since  the  latter  must  be  plugged  with  a  perforated 
rubber  stopper  at  c,  as  the  glass  funnel  limits  the  water  on 
the  inner  side.  To  prevent  too  many  germs  from  falling  from 
the  air  into  the  flask,  a  little  sterile  cotton  is  stuffed  into  the 
mouth  of  the  latter,  about  the  tube  of  the  funnel. 

As  examples,  the  preparation  of  the  three  most  frequently- 
used  nutrient  jellies  is  given  in  detail.  Other  combinations 
(e.g.,  C.  A.  G.)  are  also  to  be  recommended. 

C.  G.  Cibil's  Gelatin. — 20  gm.  Cibil's  extract  is  added  to 
1  litre  of  distilled  water,  in  which  100  gm.  gelatin  is  then 
dissolved.  Heat  till  all  is  dissolved.  Render  slightly  alka- 
line by  addition  of  sodium  carbonate.  Boil  for  ten  minutes. 


28 


Bactcriolog  ical    TccJinology. 


Cool  to  50°  0.  Clarify,  as  indicated  above.  Cook  again  for 
ten  minutes.  Filter  through  two  thicknesses  of  paper  in  the 
hot-water  funnel.  Pour  into  smaller  vessels,  and  sterilize  for 
a  short  time  in  the  steam  cylinder  on  two  or  three  successsive 
days. 

E.  P.  A.  Peptonized  Agar. — 5  gm.  Liebig's  extract;  30  gm. 
peptone;  5  gm.  cane-sugar;  15  gm.  agar;  1  litre  distilled  water. 
Cook  for  an  hour,  render  slightly  alkaline,  and  cool  to  below 
60°  C.  Clarify,  cook  again  for  at  least  an  hour,  fill  into  bottles 
or  test-tubes,  and  steam  for  ten  minutes  on  each  of  two  or 


FIG.  i5. — Plantamour  Hot-water  Funnel,  for  use  with  Gelatin  and  Agar. 


three  successive  days.  If  5  per  cent  of  sterile  glycerin  is 
added  to  this  agar,  and  the  whole  neutralized,  it  forms  the 
glycerin  B.  P.  A.  recommended  by  Roux  and  Nocard  for  the 
cultivation  of  tubercle  bacilli.  This  is  far  easier  to  prepare 
than  serum,  which  is  used  for  the  same  purpose,  cf.  infra,  p. 
29  et  seq.  and  32. 

K.  P.  A.  G.  Peptonized  Agar-Gelatin.—To  a  litre  of  fil- 
tered flesh-water  (pp.  18,  19)  add  5  gm.  table  salt,  10  gm.  pep- 
tone, 50  gm.  gelatin,  and  7.5  gm.  agar.  Treat  like  E.  P.  A.,  ex- 
cept that  it  is  to  be  cooked  each  time,  only  20  to  30  minutes. 
It  is  safest  before  final  filtration  to  filter  a  little  into  a  test- 
tube  and  see  if  it  remains  clear  after  boiling  10  to  l^minutes. 


Bacteriological    TecJinology.  29 

From  my  experiments,  jellies  made  according-  to  Mahn,  by 
the  use  of  two  per  cent  of  Cibil's  extract  (C.  G. — C.  A. — C.  A. 
G.)  afford  quite  as  good  a  medium  for  many  sorts  of  bacteria 
as  the  far  more  expensive  peptonized  flesh-water  or  meat 
gelatins,  with  or  without  sugar. 

For  the  cultivation  of  moulds  or  yeasts,  jellies  are  prepared 
by  adding  G.  or  A.  to  the  nutrient  fluids  named  above.  Espe- 
cially to  be  recommended,  are : — 

Beer- Wort  Agar  (B.  A.),  prepared  of  equal  parts  wort  and 
water,  with  1.5  per  cent  agar. 

Raisin  Gelatin  (R.  G.),  a  decoction  of  250  gm.  raisins  in  a 
litre  of  water,  to  which  is  added  10  per  cent  of  gelatin. 

3.  Serum. — Some  large  glass  jars  (e.g.,  pickle-jars — cf.  Fig. 
63)  are  tied  up  in  three  layers  of  paper,  and  sterilized  at  140°  C. 
They  are  filled  with  blood  from  oxen,  calves,  or  horses  (lambs' 
blood  is  not  to  be  recommended),  collected  during  the  slaugh- 
tering of  the  animal,  with  as  great  cleanliness  as  can  be 
obtained  in  a  slaughter  house.  Care  must  be  taken  to  avoid 
shaking  the  jars,  and  they  are  at  once  set  away  to  coagulate, 
preferably  in  cold  water.  After  this,  they  are  kept  in  a  cold 
place  (best  of  all  an  ice-box),  for  24  to  36  hours.  The  serum 
which  has  now  separated  out,  is  transferred  in  small  quanti- 
ties into  plugged  and  sterilized  test-tubes,  by  aid  of  a  pipette 
that  has  been  carefully  sterilized.  The  greatest  possible  care 
is  taken  throughout,  since  any  contamination  will  be  more 
fatal  here  than  in  preparing  the  other  culture  media,  because 
the  final  sterilization  must  be  effected  at  a  low  temperature 
and  consequently  in  an  incomplete  manner.  Wetting  the  in- 
side of  the  test-tubes  toward  the  top  with  serum  is  especially  to 
be  avoided,  as  this  part  of  the  tube  cannot  be  immersed  in  the 
water  during  the  subsequent  heating.  The  water  bath  (Fig.  4) 
is  now  filled  with  the  test-tubes  containing-  serum,  and  heated 
until  the  thermometer  indicates  58°  to  60°  C.,  at  which  tem- 
perature it  is  kept  for  a  little  over  an  hour,  by  means  of  a 
small  flame.  This  is  repeated  daily,  for  a  week.  The  serum 
treated  in  this  manner  has  become  perceptibly  clearer  than  it 
was  originally,  a  small  amount  of  whitish  precipitate  has 
separated,  and  a  thin  oily  layer  (of  cholesterin)  floats  on  the 
surface.  All  of  the  less  resistant  germs  are  destroyed  by  the 
heating. 

It  remains  to  solidify  the  serum  without  loss  of  its  trans- 


30  Bacteriological    Technology. 

parency.  Koch  effects  this  by  prolonged  heating-  at  68°  C. 
The  water  bath  is  brought  up  to  this  point  and  carefully  kept 
there  during  the  process.  According  to  the  peculiarities  of 
different  lots  of  blood,  the  time  required  for  solidifying  serum 
varies  from  one  to  several  (6  to  8)  hours,  so  that  it  is  necessary 
to  examine  a  couple  of  tubes  from  time  to  time  to  see  if  the 
coagulation  has  begun.  The  heating  is  stopped  when  it  be- 
comes easy  to  invert  the  test-tube  without  loosening  the  serum, 
which  is  now  an  amber-yellow  jelly,  in  all  cases  sufficiently 
clear  in  transmitted  light  to  permit  observation  of  the  pecu- 
liarities of  cultures  growing  along  inoculation  punctures  made 
in  it.  The  fewer  red  corpuscles  originally  present  in  the 
serum,  the  clearer  and  more  attractive  it  becomes  after  coagu- 
lation; but  use  can  be  made  of  a  somewhat  red  serum,  espe- 
cially for  cultures  on  the  surface. 

The  last  heating  at  about  70°  C.  not  only  gelatinizes  the 
serum,  but  also  naturally  contributes  to  a  complete  steriliza- 
tion; yet  one  can  onty  be  sure  of  having  killed  all  germs  by 
convincing  himself  that  the  tubes  remain  free  from  bacteria 
after  standing  for  some  time,  a  couple  of  weeks,  at  the  tem- 
perature of  the  room,  or  three  or  four  days  in  the  brood-oven 
at  30°  C.  (Cf.  Chapter  VI.) 

An  inconvenience  in  the  use  of  the  water  bath  for  steriliz- 
ing and  coagulating  serum,  already  indicated,  is  that  the  cot- 
ton plugs  and  the  upper  part  of  the  tubes,  which  project  above 
the  water,  are  not  exposed  to  so  high  a  temperature  as  the 
thermometer  shows.  This  is  avoided  by  using,  instead  of  the 
water  bath,  a  common  hot  chamber  of  the  sort  indicated  in 
Chapter  VI.,  such  as  it  is  always  necessa^  to  have  for  use  as 
a  brood-oven. 

If  it  is  desired  to  solidify  the  serum  with  a  large  oblique 
surface  (Fig.  5,  II.),  the  test-tubes  must  be  laid  in  the  chamber 
in  a  nearly  horizontal  position.  They  are  best  put  in  a  flat 
box  which  can  be  obliquely  set  in  the  brood-oven. 

Koch  uses  for  this  purpose  a  shallow,  quadrangular  ther- 
mostat covered  with  glass  and  felt.  This  is  shown  in  section  in 
Fig.  16.  The  bottom  of  this  is  30  cm.  square  (inside  measure) 
and  the  layer  of  water  between  the  double  walls  is  6  cm.  deep 
in  the  bottom  (aa)  and  3  cm.  wide  at  the  sides  (bb).  The  pro- 
jecting edge  of  the  outside  wall  (c)  rises  a  cm.  above  the  rest 
and  so  incloses  a  space  for  a  cover  consisting  of  a  square 


Bacteriological    Technology.  3 1 

plate  of  glass  (d)  (which  renders  possible  a  quick  observation 
of  the  temperature  and  the  condition  of  the  serum),  and  a 
sheet  of  felt  (e)  which  is  a  poor  conductor.  Water  is  poured 
in  through  the  tube  (/),  while  air  is  allowed  to  escape  through 
a  similar  tube  at  the  other  end.  The  apparatus  is  set  obliquely 
by  putting  a  couple  of  blocks  (g)  under  two  of  its  legs.  This 
size  accommodates  two  rows  of  test-tubes  between  which  a 
thermometer  is  laid. 

This  combination  of  "Pasteurization"  and  "disconnected 
heating,"  which  has  been  outlined  (cf.   pp.    7,    8)  first  used  by 


FIG.  16.— Shallow  Thermostat  for  Solidifying  Serum  Obliquely. 

Koch  in  preparing  serum  for  the  cultivation  of  the  tubercle 
bacillus,  must  always  be  used  when  the  blood  has  not  been 
collected  with  the  greatest  possible  cleanliness.  But  when  the 
occasion  offers  for  collecting  blood  by  skilfully  bleeding  the 
animal,  with  sterile  instruments,  after  carefully  washing  the 
neck  of  the  horse  or  cow  with  0.1  per  cent  sublimate,  it  is 
usually  safe  to  omit  the  tedious  and  difficult  preliminary  steril- 
ization and  immediately  proceed  to  solidify  it  jat  68°  to  70°  C. 
after  putting  it  into  the  test-tubes. 

If  it  is  desired  to  keep  serum  sterile  for  some  time  in  a 
large  vessel,  the  method  that  has  been  described  cannot  be 
used,  but  it  is  best  to  filter  it  through  a  Chamberland  filter 


32  Bacteriological    Technology. 

(under  low  pressure)  into  a  Pasteur  flask  (Fig-.  67),  the  tube  of 
which  is  then  quickly  sealed  by  melting-,  and  is  first  opened 
when  the  fluid  is  distributed  into  test-tubes. 

Serous  accumulations,  such  as  those  of  ascites,  pleuritis,  or 
hydrocele,  which  can  also  be  used  as  culture  media  (c/.  Chap- 
ter XL),  are  treated  exactly  like  blood  serum. 

If  6  to  8  per  cent  of  sterilized  glycerin  is  added  to  the 
fluid  serum  and  this  is  solidified  at  a  slightly  higher  tempera- 
ture than  that  which  has  been  given  (75°  to  78°  C.)  solid  gly- 
cerin serum  is  obtained,  which,  according  to  the  observation 
of  Roux  and  Nocard,  is  a  much  better  culture  medium  for  the 
tubercle  bacillus  than  serum  alone. 

Serum  can  also  be  applied  as  a  gelatinizing  constituent  of 
culture  media,  like  gelatin  or  agar.  Toeffler  has  advanta- 
geously used  a  mixture  of  three  parts  of  sterilized  fluid  serum 
and  one  part  of  neutralized  bouillon,  containing  one  per  cent 
of  peptone,  one  per  cent  of  grape  sugar  and  0.5  per  cent  of  table 
salt.  The  mixture  was  solidified  at  66°  C. 

4.  Softened  White  Bread. — White  bread  is  cut  in  slices 
and  the  soft  part  is  broken  from  the  crust  in  small  pieces, 
which  are  laid  in  a  thin  layer  under  filter  paper,  to  dry  in  the 
air.  When,  after  a  few  days,  it  is  dry  enough  to  crumble,  it 
is  finely  ground  in  a  coffee-mill,  and  this  dry  bread  powder  is 
kept  in  a  jar  covered  only  with  paper.  When  it  is  to  be  used, 
the  desired  quantity  of  the  powder  is  weighed  out,  and  poured 
into  a  sterilized  flask,  plugged  with  cotton,  so  that  it  forms  a 
level  layer  in  the  bottom  of  this  (about  8  gm.  are  used  for  one 
of  the  smallest  Erlenmeyer  flasks).  By  the  use  of  a  pipette, 
about  two  and  a  half  times  its  weight  of  sterilized  water  is 
slowly  poured  over  it.  The  bread  broth  formed  in  this  way  is 
sterilized  twice  by  heating  it  half  an  hour  in  streaming  steam, 
with  an  interval  of  a  day. 

White  bread  can  also  be  used  in  slices  which,  either  with 
or  without  previous  sterilization  by  dry  heat,  are  moistened 
with  sterile  water  and  then  sterilized  in  the  steam  cylinder. 
But  by  pulverizing  it,  a  more  uniform  and  handy  preparation 
is  obtained. 

This  moistened  bread  is  particularly  adapted  to  the  culti- 
vation of  moulds,  but  many  different  bacteria  also  thrive  upon 
it.  It  can  be  improved  and  varied  by  moistening  it  with 
bouillon,  prune  juice,  decoction  of  manure,  etc.  (Brefeld). 


Bacteriological   Technology.  33 

5.  Rice  Milk. — Very  recently,  Soyka  has  recommended  the 
following-  mixture,  which  he  has  employed  with  success  for  his 
sealed  museum  cultures  (c/.  Chapter  VI.) :  Rice  meal,  10  gm. ; 
milk,  15  cc.;  neutral  bouillon,  5  cc.,  mingled  very  thoroughly, 
filled  into  glass  trays  by  a  pipette,  and  sterilized  by  discontin- 
uous heating  in  streaming  steam   on  two  successive  days, 
during  which  it  solidifies  in  the  bottom  of  the  tray  as  a  white 
opaque  mass. 

6.  Other   Culture  Media. — Of    these,  but    three  will  be 
named:    a.  According  to  Soyka,   other  things   being-  equal, 
spore  formation  in  bacillus  anthracis  is  hastened  considerably 
by  the  addition  of  a  certain  quantity  of  sterilized  sand  to  the 
nutrient  bouillon ;  2  to  4  cc.  of  bouillon  to  25  gm.  of  sand  gives 
a  suitable  degree  of  moisture  to  this  "  artificial  soil." x 

b.  Cultivation  upon  moistened  blocks  of  plaster  was  first 
used  by  Engel  for  inducing  the  so-called  spore-formation  in 
yeasts.     Hansen  recommends  blocks  in  the  form  of  truncated 
cones,  3  cm.  high,  4  cm.  in  diameter  at  top,  and  5  cm.  at  bot- 
tom, which  are  placed  in  small  glass  trays  5  cm.  deep,  loosely 
covered  by  similar  inverted  trays.     Sterilized  water  is  poured 
into  the  trays  so  as  to  reach  to  the  middle  of  the  block.2    It  is 
also  possible  to  make  these  blocks  of  any  desired  form.     I 
have,  for  instance,  used  successfully  small  plaster  cylinders 
(moulded  in  glass  tubes),  that  were  placed  in  ordinary  cotton- 
plugged  test-tubes.     It  is  to  be  observed  that  a  mixture  of 
eight  parts  of  plaster  and  three  parts  of  water  is  used  in 
making  the  blocks.     The  mould  in  which  they  are  cast  must 
not  be  oiled.     Before  use  they  are  sterilized  by  dry  heat  of 
115°  C.,  since  a  higher  temperature  robs'  the  plaster  of  too 
much  of  its  water  of  crystallization. 

c.  Eggs  are  shaken  up  so  as  to  mix  the  white  and  yolk, 
and  the  surface  disinfected  with  sublimate  solution,  after  which 
a  s.mall  hole  is  made  through  the  shell  at  one  end,  by  use  of  a 
needle,  through  which  the  inoculation  needle  is  introduced, 
and  the  hole  closed  by  a  little  cotton  and  collodion. 

d.  Colored  nutrient  gelatin.     Noeggerath  prepares  the  fol- 
lowing- mixture  of  concentrated  aqueous  solutions  of  aniline 
dyes,  representing  approximately  the  spectral  colors:  meth- 
ylene  blue,  2  cc.;  gentian  violet,  4  cc.;  methyl  green,  1   cc.; 
chrysoidin,  4  cc.;   fuchsin,  5   cc.,  diluted  with  200  cc.  water. 
This  is  allowed  to  stand  10  to  14  days.     Of  the  blue-black  or 

3 


34  Bacteriological    Technology. 

dark  gray  fluid,  7  to  10  drops  are  added  to  each  10  cm.  of  pep- 
tonized  gelatin  (K.  P.  G.),  which  is  cooked  a  couple  of  times, 
poured  out  on  white  porcelain  plates,  and  infected  by  scratch- 
cultures  (as  in  Fig.  29).  The  bacteria  by  their  growth  produce 
color  changes  in  the  gelatin,  which  can  be  used  for  diagnostic 
purposes.3 

FILLING  THE  CULTURE  VESSELS. 

So  far  as  serum,  bread,  and  rice-milk  are  concerned,  the 
process  has  been  already  described.  Only  the  gelatinizing 
media  need  closer  consideration.  These  are  stored,  like  culture 
fluids,  in  cotton-plugged  medicine  bottles  holding  150  to  250  cc. 
each.  It  is  convenient  to  have,  in  addition  to  these, 
smaller  reservoirs,  e.g.,  large  test-tubes,  so  that 
when  only  a  little  gelatin  is  needed  it  will  not  be 
necessary  to  open  a  large  flask,  since  each  time 
that  one  is  opened,  what  remains  in  it  must  be  re- 
sterilized  for  safety. 

When  the  contents  of  a  storage  bottle  are  to  be 
distributed  into  culture  glasses  they  are  melted  in 
the  water  bath,  or  better,  the  steam  cylinder.  Test- 


d 
* 

I 
tubes  and   other  relatively   wide-mouthed  vessels          ' 

can  be  filled  by  pouring  directly  from  the  bottle, 
or  better,  by  the  use  of  large  pipettes  (Fig.  17). 
These  are  cleansed,  dried,  wrapped  in  paper,  and 
sterilized  in  the  usual  manner,  if  their  size  allows,  in 
the  dry  oven  at  150°  C.,  otherwise  they  are  drawn 
back  and  forth  for  some  minutes  through  a  gas 
or  alcohol  flame.  To  avoid  contamination  of  the 
pipettes  when  they  are  laid  down,  they  may  be  sup- 
ported on  small  knife-benches  (Fig.  18),  such  as  are 
frequently  used  by  housekeepers,  the  upper  side  of 
which  is  cleansed  by  flaming  before  they  are  used. 
The  only  other  precautions  to  be  taken  are,  to  close 
the  tubes  as  quickly  as  possible,  and  to  see  that 
each  tube  is  filled  for  at  least  3  cm.  with  gelatin, 
and  that  the  top  of  the  tube  is  not  moistened  where  it  comes 
in  contact  with  the  cotton  plug,  or  the  latter  will  become 
fastened  to  the  glass.  When  flasks  with  a  narrow  neck  are 
to  be  filled,  this  is  best  done  by  aid  of  a  common  wash-bot- 
tle, sterilized  by  steam. 


Bacteriological    Technology.  3  5 

After  filling1,  the  glasses  are  finally  sterilized  in  the  steam 
cylinder  for  five  or  ten  minutes.  If  this  apparatus  cannot  be 
used,  each  test-tube  or  flask  is  boiled  by  itself.  When  the 
gelatin  has  solidified,  the  test-tubes  are  best  kept  by  wrap- 
ping* a  few  together  in  paper,  by  which  the  surface,  and  espe- 
cially the  cotton,  is  kept  from  becoming  dusty. 

After  standing-  for  some  time,  the  g-elatin  begins  to  dry 
out,  as  is  shown  by  the  sinking  of  its  surface  at  the  middle  of 
the  tube.  When  a  needle  is  thrust  into  such  gelatin,  it  does 
not  close  after  the  needle  is  withdrawn,  but  a  crack  forms, 
which  may  materially  modify  the  appearance  of  the  culture 
when  bacteria  eventually  develop  along  the  puncture.  Before 


FIG.  18.— Pipette  on  Knife-rest. 

it  is  used,  such  gelatin  should  always  be  melted  and  allowed 
to  solidify  again. 

To  prevent  the  drying  out  of  cultures  on  gelatin  or  agar, 
which  is  especially  rapid  when  they  are  kept  at  an  elevated 
temperature,  the  cotton-plugged  ends  of  the  tubes  may  be 
covered  with  small  rubber  caps.  Care  should  be  taken  that 
both  the  caps  and  the  cotton  plugs  are  absolutely  sterile,  or 
there  is  danger  of  the  cultures  becoming  contaminated.  Mould 
spores,  especially,  which  are  unable  to  germinate  in  the  un- 
capped and  therefore  dry  plugs,  germinate  readily  in  the  moist 
cotton,  and  grow  through  it.  For  this  reason,  it  is  necessary 
to  singe  the  top  of  the  plugs,  and  to  carefully  disinfect  the 
rubber  caps  in  sublimate  immediately  before  putting  them  in 
place.  The  drying  out  of  culture  can  also  be  prevented  by 
singeing  the  cotton,  and  then  dipping  the  top  of  the  test- 
tubes  in  melted  paraffin,  which  quickly  hardens  in  an  air-tight 
layer. 


OHAPTEE  IY. 

PURE  MATERIAL  FOR  CULTURES. 

IT  has  been  shown  in  the  preceding-  chapters  that  it  is 
relatively  easy  to  sterilize  apparatus  and  nutrient  media,  as 
well  as  to  keep  them  sterile  for  an  unlimited  time.  The  only 
serious  difficulty  in  the  cultivation  of  bacteria  has  always  been 
connected  with  another  point — the  procuring-  of  pure  material 
for  starting-  the  cultures.  The  ubiquity  and  small  size  of  bac- 
teria have  necessitated  the  application  of  quite  peculiar  and 
complicated  methods.  Their  omnipresence  makes  it  hard  to 
avoid  foreign  germs,  while  their  minuteness  interferes  with 
the  sowing  of  a  single  germ,  as  is  done  in  the  cultivation  of 
higher  plants. 

For  certain  forms  of  fungi  this  may  be  attained.  Thus, 
Brefeld  long  since  (1874)  postulated  and  fulfilled  the  require- 
ments for  his  mould  cultures,  that  they  should  originate  from 
a  single  spore,  planted  under  microscopic  control;  and  in  1883 
Emil  Chr.  Hansen,  in  his  work  on  yeast,  which  is  so  full  of 
importance  for  breweries,  prepared  his  cultures,  which  demon- 
strably  start  from  one  yeast-cell,  by  the  use  of  other  and 
better  methods  than  those  used  by  Brefeld. 

On  the  other  hand,  the  evanescent,  small,  and  little  charac- 
terized germs  of  bacteria  cannot,  as  a  rule,  be  handled  in  this 
way.  It  has  been  necessary  to  seize  upon  other  means  of  get- 
ting pure  inoculation  material  for  cultures  of  these,  and  in 
1881  Koch  succeeded  in  a  simple  and  ingenious  manner  in 
overcoming  the  difficulties  and  indicating  a  way  of  isolating- 
bacteria  germs,  which  will  surely  remain  in  the  future  as  the 
chief  method. 

Before  we  proceed  to  a  description  of  Koch's  method, 
which,  with  various  modifications,  will  be  constantly  used  in 
the  work  that  follows,  we  must  briefly  consider  the  earlier 
methods  of  securing  pure  cultures,  not  only  because  of  their 


Bacteriological    Technology.  37 

historical  interest,  but  also  because  under  certain  circum- 
stances, notwithstanding  the  discovery  of  the  far  more  perfect 
process  of  Koch,  we  may  be  obliged  to  use  them,  or  may  find 
it  advantageous  to  do  so. 

All  of  the  methods  which  have  thus  far  been  used  for  se- 
curing pure  inoculation  material  may  be  arranged  in  two 
principal  groups:  A,  those  in  which  use  has  been  made  of 
physiological  differences  between  the  bacteria  for  separating 
them;  and  B,  those  in  which  the  actual  separation  of  the  germs 
from  one  another  is  used  as  the  means  of  isolation. 

A.  USE  OF  PHYSIOLOGICAL  DIFFERENCES. 

If,  for  instance,  one  virulent  form  exists  among  a  number 
of  non-parasitic  forms,  in  a  putrid  fluid,  it  may  be  obtained  in 
a  state  of  purity  by  the  inoculation  of  a  suitable  animal,  in 
the  blood  of  which  only  the  virulent  form  will  come  to  devel- 
opment while  the  others  die.  In  this  way,  from  putrid  blood, 
have  been  obtained  the  microbes  of  Pasteur's  rabbit-septicae- 
mia, Koch's  mouse-septicaemia,  and  Davaine's  rabbit-septicae- 
mia. Or,  for  another  example :  according  to  Pasteur,  in  the 
blood  of  an  animal  dead  of  splenic  fever,  which  has  been  kept 
at  a  high  temperature,  or  which,  from  the  size  of  the  animal, 
has  cooled  slowly,  there  is  found,  so  soon  as  putrefaction  has 
set  in,  besides  the  anthrax  bacillus,  that  which  he  has  called 
the  vibrion  septique  (which  is  identical  with  Koch's  bacillus 
ceclematis  maligni).  Pasteur  succeeded  in  isolating  these  two 
pathogenic  forms  by  utilizing  their  different  behavior  with 
respect  to  free  oxygen;  for  as  the  first  is  anaerobic  while  the 
second  is  aerobic,  and  both  grow  in  broth,  he  obtained  a  cul- 
ture of  one  or  the  other  by  sowing  the  blood  in  receptacles 
from  which  all  air  was  removed  and  kept  out  by  carbonic  acid 
(c/.  Chapter  VIIL),  OF  in  an  ordinary  culture  vessel.  These 
examples  might  be  increased  by  many  more,  such  as  cultures 
in  acid  and  alkaline  media,  with  or  without  the  addition  of 
certain  antiseptics,  at  higher  or  lower  temperatures,  etc.;  but 
only  two  of  the  methods  of  this  class  will  be  dwelt  upon  a 
little  more  fully,  viz. : 

1.  Klebs'  Fractional  Cultures  (1873). — A  fluid  in  which  a 
large  number  of  different  bacteria  live  and  thrive  together, 
will  necessarily  not  contain  an  equal  mixture  of  all  these 


38  Bacteriological    Technology. 

forms  at  every  point.  Some  of  the  quiescent  forms  will  sink 
to  the  bottom  or  adhere  to  the  sides,  while  others  can  move 
about  everywhere  in  the  liquid.  Some  forms  will  form  super- 
ficial pellicles,  others  thrive  only  below  the  surface,  where 
there  is  less  oxygen.  Moreover,  according1  to  the  nature  of 
the  fluid,  some  forms  will  fill  it  with  great  rapidity,  and  far 
surpass  in  numbers  others  which  only  manage  to  lead  a  re- 
stricted existence  in  the  liquid.  If  a  very  small  quantity  of 
such  a  fluid  containing  bacteria  is  sown  in  a  culture  glass,  in 
all  probability  only  a  few  forms  will  be  transferred,  and  these, 
less  equally  distributed,  will  develop  in  unequal  numbers.  If 
an  extremely  small  quantity  of  this  new  mixture,  containing 
fewer  forms,  is  again  transferred  to  a  new  culture  flask,  and 
this  process  is  repeated  several  times,  in  all  probability  a  point 
will  finally  be  reached  where  the  material  used  for  the  transfer 
is  absolute^  pure,  i.e.,  includes  only  a  single  kind  of  bacteria. 

This  method  has  only  slight  value,  and  it  is  especially  to 
be  observed  that  such  fractional  cultures  are  by  no  means  to 
be  counted  on  as  finally  giving  a  pure  culture  of  any  specified 
form  among  those  that  were  found  in  the  original  fluid,  not 
even  that  form  which  was  most  abundant  there.  The  final 
product  is  often  only  one  of  the  most  wTidely  distributed  of  the 
common  putrefactive  bacteria,  which,  even  though  originally 
present  in  small  quantity,  easily  gains  the  advantage  of  the 
others.  Still  this  method  of  fractional  cultures  may  serve  a 
useful  purpose  now  and  then,  as  supplementary  to  others. 

2.  Cohn's  Heating  Method  (1876). — It  had  long  been  known 
that  various  organic  fluids  (milk,  hay-infusion,  infusion  of 
peas,  etc.),  notwithstanding  rather  long  cooking  and  the  ex- 
clusion of  germs  from  the  air,  became  the  seat  of  bacterian 
development,  and  adherents  of  the  theory  of  spontaneous  gen- 
eration had  repeatedly  taken  this  fact  as  favoring  their  doc- 
trine; but  it  was  first  shown  by  Cohn  in  1876,  in  a  series  of 
experiments  with  hay  infusion,  that  this  must  depend  upon 
the  extraordinary  resistance  of  certain  bacillus-spores  to  heat. 
The  boiling  heat  rapidly  destroyed  all  bacteria  without  spores, 
but  the  latter  (in  Cohn's  experiments  those  of  bacillus  sub- 
tilis)  where  not  killed — a  fact  which  Brefeld  also  showed  later 
by  direct  microscopic  observation  of  the  germination  of  the 
cooked  spores.  This  afforded  a  means  of  obtaining  pure  ma- 
terial of  certain  spore-forming  bacilli,  and,  as  a  rule,  a  pure 


Bacteriological    Technology.  39 

culture  of  bacillus  subtilis  can  be  obtained  without  difficulty 
in  the  following1  manner:  A  small  quantity  of  water  is  poured 
over  some  hay  in  a  flask,  which  is  kept  for  hours  at  30°  to  40° 
C.  (either  in  the  brood-oven,  or  directly  over  the  flame,  while  a 
thermometer  is  placed  in  the  fluid).  The  dark  reddish-brown 
infusion  is  diluted  with  distilled  wrater  until  it  reaches  a  clear 
golden  color  (s.  g.  1.006),  when  it  is  filtered  through  muslin, 
neutralized  with  sodium  carbonate,  distributed  in  several  ves- 
sels plugged  with  cotton,  and  cooked  for  ten  minutes,  after 
which  it  is  set  away  in  the  brood-oven  at  30°  to  40°  C.  In  the 
course  of  a  couple  of  days,  a  larger  or  smaller  number  of  the 
vessels  will  contain  a  growth  of  bacillus  subtilis,  which  may 
be  known  by  the  fact  that  it  forms  a  firm  rigid  film  on  the 
surface  of  the  liquid. 

It  must  be  remembered  that  among-  the  various  resistant 
spores  of  bacilli  there  is  a  great  difference  in  their  ability  to 
endure  heat  of  100°  C.  Some  survive  only  a  very  few  moments 
of  cooking,  while  others  bear  this  temperature  for  hours  (cf. 
Chapter  XIL).  The  limitations  of  this  method  are  indicated 
by  what  has  been  said.  It  is  possible  to  isolate  by  it  only 
those  bacilli  which  form  very  resistant  spores,  and  a  really 
pure  culture  is  obtained  with  certainty  only  when  the  boiled 
fluid  does  not  contain  several  different  forms  with  the  same 
power  of  resistance.  Still  there  will  be  exceptional  cases  in 
which  there  is  use  for  cooking  as  an  aid  in  isolation. 

B.  METHODS  BY  SEPARATION. 

1.  The  Capillary-tube  Method  (Salomonsen,  1876).— Greater 
success  has  been  reached  by  the  use  of  methods  in  which  an 
actual  separation  of  the  original  germs  is  secured.  The  oldest 
of  these  is  the  following-,  the  starting-  point  for  which  was  an 
observation  of  the  color  changes  in  putrefying-  blood.  During 
decomposition,  bright  red  ox  blood,  defibrinated  by  whipping-, 
assumes  a  dark  red  or  red-brown  color,  which  is  partly  due  to 
the  removal  of  their  color  from  the  red  corpuscles,  partly  to 
deoxidation  of  the  oxyhgemoglobin,  and  partly  to  other  chemi- 
cal processes.  If  the  blood,  immediately  after  being  defibrin- 
ated, is  set  away  in  a  cylindrical  glass  where  it  can  remain 
entirely  undisturbed  at  a  comparatively  low  temperature,  e.g., 
10°  C.,  it  is  seen  that  the  change  of  color  begins  in  spots  here 
and  there  in  the  mass  of  blood.  A  closer  investigation  shows 


Bacteriological    Technology. 


that  these  putrefactive  blotches  are  due  to  bacteria  (or  moulds 
or  yeasts).  Next  the  bottom  (Fig1.  19),  where  the  crowded 
blood  discs  form  a  solid  mass,  in  which  the  bacteria  grow 
equally  in  all  directions,  the  spots  are  round,  clearly  denned, 
and  dark  red.  Above  (6)  they  are  elongated,  often  club- 
shaped,  less  clearly  outlined,  and  by  no  means  of  so  dark  a 
color.  Here  the  blood  discs  are  suspended 
in  a  relatively  large  quantity  of  serum, 
and  in  this  fluid  the  bacteria  have  been 
able  to  sink  uninterruptedly  to  the  bottom, 
marking  their  path  by  elongated  spots  or 
streaks. 

If  this  is  to  be  utilized  in  obtaining  pure 
material  of  the  various  putrefactive  bac- 
teria which  grow  in  blood,  it  is  only  neces- 
sary to  draw  the  defibrinated  ox  blood  into 
long  capillary  glass  tubes  (50  to  60  cm. 
long  and  0.5  to  1  mm.  in  diameter),  and  to 
attach  these  to  strips  of  card-board  about 
3.5  cm.  wide,  by  means  of  a  drop  of  varnish  FIG.  19.  —  Cylindrical 
at  each  end,  which  at  the  same  time  seals  Glass  containing  whipped 

'  .  Ox  Blood  showing  Purifl- 

them.     The  following  can  be  recommended  cation  Blotches.  a,serum 
as  a   suitable  cement:   8   parts  of    rosin,  layer;  6,  elongated  disooi- 

orations;    c,    round    spots 

melted  with  2.5  parts  of  wax  while  the  and  precipitated  biood- 
mass  is  constantly  agitated.  Of  1  part  of  corpuscies. 
turpentine,  enough  is  added,  little  by  little,  so  that  a  drop  of 
the  melted  mass  quickly  solidifies  when  allowed  to  fall  on  a 
glass  plate.  Another  good  cement  is  the  "Cire  Galoz"  (to 
be  had  of  Alvergniat  Freres,  10  Rue  de  la  Sorbonne,  Paris). 

They  are  then  laid  away  at  the  ordinary  temperature  of 
the  room,  for  daily  observation.  The  putrefaction  blotches 
will  soon  appear ;  some  shortly  after  the  drawing  of  the  blood, 
others  many  days  later;  some  spreading  with  extraordinary 
rapidity,  others  growing  only  slowly.  The  number  of  each 
spot  in  sequence,  the  time  of  its  appearance,  as  well  as  its 
growth  from  day  to  day,  are  easily  noted  upon  the  card.  The 
growth  is  best  indicated  by  pencil  marks  drawn  every  morn- 
ing and  night  after  the  fashion  shown  in  Fig.  20.  This  illus- 
tration, which  shows  one  end  of  a  card  to  which  is  cemented 
a  capillary  tube  containing  three  putrefactive  spots,  renders 
further  description  unnecessary. 


Bacteriological    TccJmology.  41 

These  putrefaction  spots  come  from  the  bacterian  germs 
which  have  chanced  to  get  in  the  blood  after  its  removal  from 
the  animal,  so  that  in  one  lot  there  may  be  many  of  them,  in 
another  few;  but  each  spot  contains  only  a  single  sort  of  bac- 
teria, developed  from  one  germ.  Consequently,  when  the  clis- 
colorations  are  relatively  far  apart  in  the  tubes  so  that  they 
do  not  readily  become  confluent,  each  of  them  gives  a  small 
pure  culture,  from  which  pure  material  may  be  obtained  of  a 
specific  form  of  bacteria.  By  this  method,  a  mixed  putrefac- 
tion-flora was  first  separated  into  its  elements,  so  that  the 
number  of  germs,  the  time  of  their  development,  rapidrty  of 
growth,  etc.,  could  be  graphically  represented. 

2.  By  Dilution  (Lister,  1878,  Naegeli,  1879).— This  method 
consists  in  diluting  a  fluid  containing  bacteria  with  sterile 


FIG.  20.— One  end  of  a  Pasteboard  Bearing  a  Capillary  Tube  Filled  with  Blood.  On  the 
card  are  noted  the  time  (10  A.— afternoon  of  10th  day;  9  M.— morning  of  the  9th  day),  'when 
the  spots  appeared,  the  rapidity  of  their  growth  (indicated  by  the  length  of  the  lines),  and 
their  number  in  series  (/.,  //.,  V.) 

water  to  such  a  degree  that  a  given  quantity  of  the  mixture 
holds  only  erne  germ,  and  in  using  such  an  initial  quantity 
for  inoculation,  In  the  culture-flask,  the  offspring  of  this 
single  germ  forms  a  pure  culture. 

Counting  the  germs  in  the  original  fluid  is  effected  under 
the  microscope,  by  the  aid  of  instruments  similar  to  those 
used  for  counting  blood-discs.  From  this  the  number  of 
germs  in,  for  example,  0.05  cc.  of  the  fluid,  is  estimated,  and  by 
aid  of  a  sterilized  graduated  pipette,  this  quantity  is  added  to 
enough  sterile  water  so  that  the  mixture,  after  careful  shak- 
ing, must  average  half  a  germ  for  each  0.05  cc.  When  by 
means  of  a  sterilized  graduated  pipette,  this  mixture  is  inocu- 
lated into  a  series  of  culture-vessels,  so  that  0.05  cc.,  is  placed 
in  each,  half  of  the  glasses  will  not  show  any  development  of 
bacteria,  having  received  no  germs,  while  in  all  probability 
but  one  germ  will  have  been  placed  in  each  of  the  others, 
which  will  then  contain  a  pure  culture.  Lister  devised  and 


42  Bacteriological    Technology. 

used  this  method  for  obtaining-  pure  cultures  of  lactic  acid 
bacilli.  Naegeli,  independently,  cultivated  micrococcus  ureas 
by  aid  of  the  method  of  dilution. 

3.  By  the  Use  of  Solid  (Especially  Gelatinized)  Media 
(Koch,  1881). — This  method  consists  in  the  isolation  of  the 
germs  upon  or  in  solid  culture-media,  where,  in  their  later  de- 
velopment, they  give  rise  to  separated  colonies.  If,  in  a  flask 
like  that  shown  in  Fig-.  9,  VII.,  the  bottom  is  covered  with  a 
thin  layer  of  nutrient  g-elatin,  and,  after  melting  this  at  about 
30°  C.,  a  very  small  drop  of  fluid  containing  bacteria  is  added 
and  thoroughly  distributed  through  the  gelatin  by  careful 
shaking  (cf.  Chapter  VIL),  and  after  solidifying,  the  mixture 
is  set  aside  at  room  temperature,  after  a  time  small  colonies 
will  appear  at  the  surface  of  the  gelatin.  Each  comes  from 
one  germ,  so  that  pure  material  for  inoculation  is  obtained. 

This  is  but  one  of  the  many  forms  in  which  the  method  of 
Koch  may  be  used.  In  a  subsequent  chapter,  dealing  with 
bacteriological  analysis,  the  method  will  be  treated  particu- 
larly and  in  detail. 

By  aid  of  these  methods,  pure  inoculation  material  can 
comparatively  easily  be  obtained  of  all  aerobic  bacteria  (cf. 
Chapter  VIII.)  which  it  is  chiefly  necessary  to  cultivate.  It 
remains  only  to  consider  the  best  way  of  transferring  this  to 
the  culture  apparatus. 


CHAPTER  V. 

INOCULATING  CULTURES. 

THE  transfer  of  pure  material  to  one  of  the  culture  vessels 
that  have  been  described,  is  comparatively  easy.  The  neces- 
sary opening-  of  the  different  vessels,  and  the  transportation 
of  the  inoculation  material  through  the  air  necessarily  entails 
danger  of  contamination.  But  this  danger  is  very  small,  and 
if  the  work  is  done  rapidly  and  carefully,  the  transfer  is  un- 
successful in  a  very  insignificant  number  of  cases. 

The  instruments  needed  are:  1,  Platinum  needles  (Koch), 
long  capillary  tubes  of  glass,  Pasteur  pipettes,  and  glass 
needles. 

1.  The  platinum  needle  consists  of  a  glass  rod  about  27 
cm.  long  and  4  or  5  mm.  thick,  in  one  end  of  which  a  piece  of 
platinum  wire  about  3  cm.  long  is  melted  as  shown  in  Fig.  21. 
Just  before  use,  the  surface  of  the  glass  rod  is  flamed,  and  the 
platinum  wire  brought  to  redness  throughout  its  entire  length. 
When,  after  a  few  seconds,  it  has  cooled  sufficiently,  this  is 
brought  in  contact  with  the  material  to  be  transferred,  and 
quickly  brought  into  the  culture  medium. 

It  is  usual  to  have  at  hand  some  quite  thin  needles,  and 
some  which  are  a  little  thicker.  An  advantage  of  the  former 
is  that  they  cool  quickly,  while  the  latter  are  less  flexible  and 
can  for  this  reason  be  used  when  the  needle  is  to  be  thrust 
into  more  resistant  substances,  e.g.,  liver  or  lung  (or  when  it 
is  necessary  to  apply  considerable  lateral  pressure,  as  in  dis- 
tributing a  colony  of  the  tubercle  bacillus  over  agar,  etc.). 

For  certain  cases,  the  platinum  wire  may  be  specially 
shaped.  Small  loops  (Fig.  21,  b)  insure  the  adhesion  of  larger 
quantities  of  the  inoculation  material.  A  large  loop  (c), 
catches  a  good-sized  drop.  It  is  bent  at  a  right  angle  (d), 
when  one  wishes  to  scratch  the  material  into  the  surface  of  the 
gelatin  (cf.  what  is  said  elsewhere  about  cultures  on  potato, 
slides,  etc.). 


44 


Bacteriological    Technology. 


When  the  inoculation  must  occur  through  a  relatively 
small  opening-,  through  which  the 
glass  rod  can  be  passed  with  diffi- 
culty or  not  at  all,  a  sufficiently  long- 
piece  of  platinum  wire  can  be  used, 
or  small  pieces  (5  or  6  mm.  long)  are 
employed  by  seizing  them  with  ster- 
ilized forceps,  glowing-  them,  bringing 
them  in  contact  with  the  inoculation 
material,  and  dropping  them  through 
the  small  opening-  into  the  culture 
flask.  Capillary  tubes  and  glass  nee- 
dles can,  of  course,  also  be  used  in 
such  cases. 

2.  Capillary  tubes  usually  have  a 
length  of  20-25  cm.  and  are  closed  by 
melting  at  both  ends.     Before  being 
used,  one  end  is  broken  off,  the  surface 
is  flamed,  and  the  open  end  dipped  into 
the  fluid  to  be  sown,  when  the  other 
end  is  broken,  upon  which   the  fluid 
rises  into  the  tube.     The  contents  are 
blown  out  into   the  culture-vessel  to 
be  used ;  care  being-  taken  not  to  blow 
all  out,  but  to  leave  a  little  fluid  in  the 
tube,  thus  preventing-  contamination 
from  the  air  blown  in. 

3.  Pasteur  pipettes  consist,  as  is 
shown  in  Fig.  22,  of  a  piece  of  glass 
tubing,  one  end  of  which  is  closed  by 
a  cotton  plug,  which  must  not  project 
be3^ond  the  glass,  while  the  other  end 
is  drawn  out  into   a   capillary  tube 
fused  at  the  end.     The  plugged  tubes 
are  sterilized  at  150°  C.    Before  being 
used,  the   sealed   end   of  the  tube  is 
broken  off,  and  this  part  of  the  glass 
is  flamed.     The  tube  is  filled  by  suc- 
tion, and  emptied  by  bio  wing  the  fluid 
out.     These  pipettes  have  the  advan- 
tage over  capillary  tubes  of  being  more  capacious,  and  they 


Bacteriological    TecJinology.  45 

can  be  used  as  culture-glasses  by  reclosing  the  capillary 
extremity  by  heat  immediately  after  filling-.  See  also  Chap- 
ter XL 

4.  Glass  needles,  i.e.,  glass  rods  drawn  out  into  a  very 
slender  thread  at  one  end  for  about  15  cm.,  are  better  than 
platinum  needles  in  their  perfect  smoothness  and  rigidity, 
which  may  occasionally  be  of  importance,  as  in  making-  thrust- 
cultures  of  anaerobic  forms,  where  it  is  wished  to  avoid  intro- 
ducing small  air-bubbles  into  the  gelatin. 

One  can  easily  make  these  inoculation  instruments  for 
himself  by  using-  an  ordinary  Bunsen  burner  or  a  good  spirit 
lamp.  A  glass-blower's  outfit  naturally  lessens  the  work,  but 
it  is  not  necessary. 

For  platinum  needles,  a  glass  rod  about  4  mm.  in  diameter 
is  divided  into  pieces  25  or  30  cm.  long,  by  slightly  marking-  it 
at  the  proper  points  with  a  triangular  file,  when  by  steadily 
pulling-  the  glass  in  opposite  directions  at  each  side  of  the 
scratch,  with  a  slight  side  motion,  it  is  easily  and  evenly 
broken  at  the  desired  spot.  The  end  of  a  piece  into  which  the 
needle  is  to  be  fastened  is  rotated  in  the  flame  with  the  left 
hand,  until  it  becomes  red  and  soft,  while  a  piece  of  wire  of 
the  right  length,  grasped  some  5  mm.  from  one  end  in  a  pair 
of  forceps,  is  brought  to  a  white  heat  at  this  end  and  carefully 
thrust  lengthwise  into  the  softened  glass,  which  is  then  al- 
lowed to  cool  gradually,  being  held  for  a  few  seconds  close  to 
the  flame.  The  other  end  of  the  glass  is  finally  rounded  off 
by  heating  it  to  redness,  rotating-  it  meantime. 

A  glass  tube  6  or  8  mm.  in  diameter  is  easily  drawn  into 
capillary  tubes  in  the  following  manner :  The  tube  is  heated 
to  redness  for  a  short  distance  (1-2  cm.),  while  being  rapidly 
revolved  about  its  axis,  until  it  becomes  thoroughly  softened, 
when  it  is  removed  from  the  flame  and  drawn  into  a  tube 
about  six  feet  long,  which  is  melted  off  from  the  thicker  glass 
at  each  end.  In  the  same  way,  by  aid  of  the  flame,  the  long 
capillary  tube  is  divided  into  pieces  of  the  length  indicated 
above,  which  are  at  the  same  time  hermetically  sealed  at  the 
ends.  The  strong  heating  of  the  glass,  and  the  sealing  of  the 
tube  which  immediately  follows,  insure  freedom  from  germs. 
The  larger  the  original  tube  the  greater  the  length  for  which 
it  is  melted,  and  the  slower  it  is  drawn  out  or  the  shorter  the 
length  of  the  capillary  tube  the  coarser  this  will  be,  and  con- 


Bacteriological    Technology. 


versely.     This  being-  borne  in  mind,  it  is  easy  to  experiment 
until  a  tube  of  the  diameter  wished  in  a  given  case  is  obtained. 

Pasteur  pipettes  are  made  by  dividing  a  glass  tube  into 
pieces  15  cm.  long,  by  aid  of  a  file.  Each  piece  is  then  heated 
in  the  middle  while  being  revolved,  and  drawn  out  in  the 
manner  already  described  into  a  capillary  portion  about  30  cm. 
long,  which  is  then  melted  and  sealed  at  the  middle,  making 
two  pipettes.  The  sharp  edges  of  the  other  ends  are  rounded 
off  by  briefly  glowing  in  the  flame.  After  they  are  cooled, 
they  are  plugged  with  cotton,  and  finally  sterilized  at  150°  C. 

Glass  needles  are  drawn  out  in  the 
same  way,  solid  rods  being  used  instead 
of  tubing. 

To  indicate  the  many  little  manip- 
ulations necessary  in  making  a  pure 
transfer,  a  detailed  account  is  here  given 
of  (a)  the  inoculation  of  g-elatin  in  a 
test-tube  by  use  of  the  needle,  and  (b) 
the  inoculation  of  fluid  in  a  narrow- 
necked  flask,  by  the  use  of  small  pieces 
of  wire. 

a.  Several  tubes  of  gelatin  that  has 
not  dried  out  too  much  (cf.  p.  474)  are 
chosen,  and  the  cotton  plugs  are  tested 
by  twisting  them  several  times  to  be 
sure  that  they  are  not  glued  fast  to  the 
glass.  The  needle  is  sterilized  as  indi- 
cated above.  After  allowing  it  to  cool 
for  a  couple  of  minutes,  the  tube  from  which  the  transfer  is  to 
be  made  is  opened  by  removing  the  plug  by  a  twisting  motion 
which  causes  any  fibres  that  may  have  adhered  to  the  glass  to 
lie  out  of  the  way  of  the  needle.  The  tube  is  held  with  the  top 
upward,  or  inverted,  according  as  the  bacteria  growing  in  it 
have  liquefied  the  gelatin  or  not.  The  removed  plug  is  held 
so  that  the  part  of  it  which  comes  in  contact  with  the  inner 
face  of  the  glass  shall  not  touch  anything  by  which  it  might 
be  contaminated.  The  needle  is  now  quickly  plunged  into  the 
culture,  removed,  and  the  plug  replaced.  (As  a  rule  only  the 
tip  of  the  needle  is  brought  in  contact  with  the  culture,  a  suffi- 
cient number  of  germs  will  always  adhere  to  it.  If  for  any  rea- 
son a  large  quantity  is  to  be  transferred,  loops  such  as  are 


FIG.  23.— Inoculating  a  Test- 
tube  of  Gelatin. 


Bacteriological    Technology.  47 

shown  in  Fig-.  22,  b  c  are  used.)  The  tube  to  be  inoculated, 
held  in  an  inverted  position  (Fig1.  23),  is  now  quickly  opened 
with  the  same  twisting-  motion,  the  needle  is  rapidly  thrust 
into  the  gelatin  at  one  or  more  points,  the  plug*  replaced, 
and  the  needle  at  once  sterilized  by  glowing.  A  label  contain- 
ing- record  of  date,  source  of  material  used,  etc.,  is  then  pasted 
upon  the  tube. 

b.  The  surface  of  the  flask  is  carefully  dried,  after  which 
the  lower  part  of  the  rubber  tube  and  the  contiguous  part  of 
the  neck  are  flamed  by  use  of  a  burner  or  alcohol  lamp,  and 
the  rubber  is  slipped  up  a  little,  so  that  it  can  easily  be  re- 
moved by  one  hand.  Sterilized  forceps  are  placed  upon  glass 
benches  (Fig-.  18)  or  in  a  shallow  glass.  Small  pieces  of  plati- 
num wire  are  kept  ready  in  a  glass  tray.  One  of  these  is 
grasped  by  one  end  with  the  forceps,  at  right  angles  to  its 
axis.  The  wire  is  broug-ht  to  a  red  heat,  allowed  to  cool,  and 
plunged  into  the  colony  of  bacteria,  etc.,  from  which  the  trans- 
fer is  to  be  made.  With  the  left  hand,  the  flask  is  now  quickly 
opened,  the  wire  dropped  into  the  narrow  opening,  the  cap 
firmly  replaced,  and  a  label  prepared. 


OHAPTEE  VI. 

BROOD-OVENS  AND  THERMO-REGULATORS. 

A  MAJORITY  of  the  bacteria  known  and  cultivated  up  to  the 
present  time  thrive  at  the  ordinary  temperature  of  a  room 
(15°  to  20°  C.).  Consequently  if  it  is  only  desired  to  keep  cul- 
tures going1  and  to  observe  their  growth,  it  usually  suffices  to 
set  them  aside  in  a  living  room.  For  preserving  cultures  for 
a  long  time,  especially  as  museum  specimens,  Soyka  recom- 
mends inoculating  solid  media  in  the  small  glass  boxes  shown 
in  Figure  11,  which  are  then  carefully  sealed  with  paraffin.  By 
this  means  he  succeeds  in  checking  the  growth  of  the  colony 
at  a  certain  point  of  its  development,  while  the  capability  of 
germination  is  preserved,  and  drying  out  is  hindered.  For 
details  see  "Zeitschr.  f.  Hygiene/'  1888.  After  a  shorter  or 
longer  incubation  period,  they  will  be  seen  to  start  into 
growth,  and  even  by  the  naked  eye  it  is  possible  to  observe 
the  great  dissimilarity  which  often  exists  between  cultures  of 
different  bacteria.  Even  in  1880,  when  fluid  cultures  were 
almost  exclusively  used,  I  noticed  in  detail  the  microscopic 
differences  that  cultures  often  present,  and  called  attention 
to  their  indications  in  judging  of  the  purity  of  a  culture,  and 
the  diagnosis  of  bacteria,  which  was  sometimes  more,  easily 
made  by  the  naked  eye  than  microscopically.  When,  later, 
Koch's  gelatin  cultures  came  into  use,  still  more  occasion 
was  offered  for  the  observation  of  evident  microscopic  differ- 
ences between  cultures  of  bacteria,  such,  for  instance,  as  are 
shown  in  Figures  42  and  69.  Sometimes  it  is  very  necessary 
to  cultivate  bacteria  at  a  higher  and  more  uniform  tempera- 
ture than  that  usual  in  our  living  rooms,  for  some  forms  de- 
velop quickly  only  at  higher  temperatures,  others  never  thrive 
below  30°  C.,  and  some  species  demand  heat  for  the  produc- 
tion of  spores.  Moreover,  indeed,  as  Pasteur  first  showed, 
cultivation  at  a  high  temperature  may  change  the  physiolog- 


Bacteriological    Technology. 


49 


ical  character  of  bacteria,  and  is  used  in  the  fabrication  of 
"  vaccines."  It  may  also  be  frequently  desirable  to  cultivate 
a  micro-organism  for  a  long"  time  at  a  constant  temperature, 
whatever  this  may  be. 

Hence  are  used  thermostats  or  brood-ovens,  in  which  the 
temperature  is  held  unchanged  for  weeks  or  months  at  the  same 


FIG.  24.— Brood-oven  Covered  with  Felt,  and  Controlled  by  a  Thenno-regulator. 

point.  The  maintenance  of  a  constant  temperature  is  beset 
with  certain  difficulties.  The  changes  in  temperature  of  the 
room  in  which  the  thermostat  stands  is  disturbing1;  to  coun- 
teract them  the  brood-oven  is  inclosed  in  as  thick  a  layer  of 
some  non-conducting-  material  as  possible,  and  set  up  in  a  cellar 
or  similar  place,  where  the  temperature  changes  but  little. 
Variations  in  the  strength  of  the  source  of  heat  used  also  act 
in  the  same  way,  as  is  especially  evident  when  gas  is  used, 
4 


50  Bacteriological    Technology. 

since  the  gas  pressure  may  vary  much  in  the  course  of  the  day. 
This  may  be  obviated  by  the  use  of  pressure-regulators  and 
thermo-regulators  of  various  construction. 

A  serviceable  small  thermostat,  comparatively  cheap,  and 
self-regulating,  is  shown  in  Figure  24.  It  is  a  quadrangular 
box  of  zinc  or -copper,  set  upon  a  strap-iron  support  with  legs 
about  24  cm.  high.  The  top  and  bottom,  as  well  as  three  of 
the  side  walls  are  double,  and  the  space  between  them,  which 
is  2  to  2.5  cm.  wide,  can  be  entirely  filled  with  water  through 
the  opening  (a),  which  is  afterward  closed  by  <a  cork.  The 
fourth  side  is  single,  and  serves  as  a  door..  In  the  centre  of 
the  top,  an  open  tube  3  cm.  in  diameter  is  fastened  into  the 
wall,  piercing  it.  Into  this  by  aid  of  a  perforated  cork,  a  ther- 
mometer (b)  is  set,  which  projecting  into  the  interior  of  the 
box,  can  be  examined  without  the  necessity  of  opening  the 
thermostat.  Usually  a  thermometer  is  also  kept  entirety  in 
the  thermostat,  standing  in  a  vessel  filled  with  oil,  which 
prevents  the  mercury  from  sinking  too  rapidly  when  the  glass 
is  removed  to  observe  the  temperature.  On  the  inner  face  of 
the  two  side  walls  are  fastened  a  number  of  small  ledges  (cc) 
on  which  glass  or  tin  shelves  may  be  laid  to  provide  support 
for  a  large  number  of  low  objects.  The  outside  of  the  box  is 
covered  with  felt,  and  provided  with  a  glass  tube  for  showing1 
the  height  of  the  water  (d),  and  a  faucet  (e).  It  is  heated  b}T 
a  Bunsen  burner,  the  tube  of  which  has  been  removed,  so  that 
it  gives  a  pointed  white  flame,  which  permits  the  flame  to  be 
reduced  to  a  minimum  with  no  danger  that  it  will  "snap 
back."  [If  the  brood-oven  is  to  be  kept  but  little  above  the 
room  temperature,  there  is  some  danger  that  the  flame  may 
occasionally  go  out,  allowing  the  escape  of  a  large  quantity 
of  gas  into  the  room  before  it  is  discovered.  For  this  reason, 
the  safety  burner  devised  by  Koch,  and  to  be  had  of  dealers 
in  bacteriological  apparatus,  though  somewhat  expensive,  is 
to  be  strongly  recommended,  since  it  promptly  and  automati- 
cally cuts  off  the  supply  of  gas  in  case  such  an  accident 
occurs. — W.  T.]  The  temperature  is  held  at  a  constant  point 
by  the  use  of  the  regulator  (g),  which  is  passed  into  the  water- 
filled  space  through  an  opening  at  b.  Two  generally  used  and 
good  types  of  thermo-regulator  are  shown  in  Figures  25  and 
26,  Rohrbeck's  (Fig.  25)  is  especially  to  be  recommended. 

Reichert's  latest  regulator  is  shown  in  Figure  26.     The  gas 


Bacteriological    Technology. 


enters  at  a,  passes  through  the  opening-  b,  and  out  toward  the 
burner  at  c.  The  column  of  mercury  is  adjustable  by  the 
screw  d.  In  case  this  should  rise  so  as  to  entirely  close  the 
opening-  &,  the  flame  is  not  extinguished,  for  a  minute  opening- 
is  provided  in  the  T-shaped  tube  at  e,  which  permits  the  pas- 
sage of  just  enough  gas  to  keep  the  flame  alive,  without  heat- 
ing the  thermostat  appreciably.  The  right  size  of  this  pin- 


FIG.  25.  — Rohrbeck's  FIG.  26.-  Reichert's 
Thermo-regulater.      Thermo-regulator. 


FIG.  27.— Bohr's  Thermo-regulator. 


hole  is  reached  when  the  gas  passing  through  it  burns  with  a 
perfectly  blue  flame.  .   . 

Rohrbeck's  modification  of  the  Lothar-Meyer  regulator,  is 
shown  in  Fig.  25.  It  is  much  more  sensitive  than  the  Reicherfc 
instrument,  since  it  is  regulated  by  mercury  and  ether,  the 
vapor  of  which  changes  in  tension  comparatively  strongly 
with  slight  changes  of  temperature.  The  gas  enters  at  a, 
traverses  the  tube  d,  in  which  at  the  bottom  an  acute,  sharp- 


52  Bacteriological    Technology. 

angled  triangular  opening  (b)  is  made,  and  passes  out  toward 
the  burner  at  c.  The  mercury  is  indicated  by  line-shading, 
vapor  of  ether  fills  the  chamber  cj  [which  is  limited  by  a  glass 
diaphragm  hi,  prolonged  downward  into  a  tube  open  at  the 
bottom,  which  plunges  well  into  the  mercury].  When  the  ap- 
paratus is  warmed,  the  mercury  is  forced  by  the  expanding 
ether-vapor  into  the  tube  hh,  say  to  the  dotted  line  i,  so  as  to 
partly  close  the  triangular  opening,  which  is  then  enlarged 
or  diminished  according  as  the  mercury  falls  orrises.  From 
the  form  of  the  opening,  and  its -sharp  angles,  this  adjustment 
occurs  very  uniformly.  The  first  adjustment  of  the  apparatus 
is  effected  by  sliding  the  tube  d  up  or  down  through  the  bored 
cork  by  which  it  is  adapted  to  the  larger  tube.  Fig.  25  show 
the  regulator  in  its  cheapest  fprm.  There  is  also  a  more  im- 
proved form,  in  which  the  tube  d  is  of  steel,  and  adjustable  by 
a  fine  screw,  (Fig.  24,  g). 

A  regulator  constructed  by  Bohr  (Fig.  27),  offers  consider- 
able advantages  as  compared  with  these  commonly  used 
models.  The  reservoir  a,  filled  with  air,  is  brought,  with  its 
stopcock  b  open,  into  the  chamber  that  is  to  be  kept  under 
control.  Shortly  before  the  desired  temperature  is  reached, 
the  cock  b  is  closed,  after  which  every  rise  of  temperature, 
however  small,  will  cause  an  expansion  of  the  air  in  a  and  a 
displacement  of  the  column  of  mercury,  which  closes  the  open- 
ing d,  allowing  the  gas  to  pass  only  through  the  reserve  open- 
ing e.  It  must  be  seen  that  the  inside  of  the  reservoir  a  is 
not  damp  when  the  apparatus  is  put  in  use.  Enough  mercury 
is  poured  in  through  /  to  reach  as  high  as  c7,  so  that  this  open- 
ing is  entirely  closed  by  a  slight  displacement  of  the  mercury. 
It  is  advantageous  to  have  the  U-shaped  tube  constricted  at  c. 

In  addition  to  its  great  simplicity,  the  Bohr  regulator  is 
superior  to  those  previously  described  in  that  the  same  in- 
strument can  be  used  at  any  desired  temperature  below  the 
melting-point  of  the  glass,  and  is  adjusted  for  any  tempera- 
ture with  extreme  ease.  It  is  also  equally  sensitive  for  all 
temperatures.  The  influence  that  considerable  barometric 
changes  exert  on  it  (as  well  as  on  the  Rohrbeck  model)  is 
readily  compensated  by  opening  the  cock  (b)  for  a  moment. 
It  further  deserves  mention,  that  by  Bohr's  regulator  a  con- 
stant mean  temperature  can  be  maintained  in  larger  spaces, 
since  the  reservoir  a  may  be  given  any  desired  form  and  size; 


Bacteriological   Technology.  53 

or  a  long-  lead  tube  with  one  end  hermetically  sealed  (pinched 
or  melted  together)  may  be  used  as  a  reservoir,  passing-  into 
various  parts  of  the  chamber. 

A  thermostat  of  this  construction,  with  regulator,  will  gen- 
erally be  sufficient  even  for  finer  experiments,  which  demand 
a  constant  temperature  during  months.  In  most  cases,  how- 
ever, we  need  only  to  have  a  chamber  in  which  bacteria  can 
be  grown  at  something  above  30°  C.,  and  for  this  a  thermo- 
stat is  perfectly  good  and  useful,  even  if  it  varies  a  few  degrees 
in  the  course  of  the  day.  If  too  much  is  not  required,  the  reg-- 
ulator  may  be  entirely  dispensed  with,  and  an  ordinary  gas, 
petroleum,  spirit,  or  oil  flame  used.  Of  these  four  sources  of 
heat,  the  first  and  last  are  unconditionally  to  be  recommended. 
In  case  the  apparatus  is  to  be  kept  in  our  working  room, 
petroleum  is  less  satisfactory  because  of  its  ill-odor,  independ- 
ently of  the  greater  danger  from  fire.  Alcohol  is  better  than 
[lard]-oil  only  in  giving  a  flame  free  from  smoke,  while  oil  is 
cheaper  and  requires  less  care  in  its  use. 

It  is  naturally  most  convenient  to  use  gas  when  this  is  at 
hand  in  the  room.  Since  the  gas  flame  as  a  rule  needs  to  be 
only  small,  when  a  small  thermostat  standing-  in  an  inhabited 
room  is  to  be  kept  at  30°  to  40°  C.,  a  pointed  yellow  flame  is 
always  to  be  used  (c/.  p.  489).  By  a  few  days'  experimenting, 
the  proper  height  of  the  flame  is  ascertained,  and  when  the 
temperature  of  the  surrounding  air  does  not  vary  too  much 
this  affords  a  good  guide.  A  regular  periodical  change  in  the 
gas  pressure,  as  at  night,  is  easily  compensated  for  by  slightly 
raising-  or  lowering  the  burner  each  day  at  the  necessary 
time. 

If  one  is  unable  or  unwilling  to  use  gas,  [lard]- oil  is  to  be 
recommended,  especially  if  used  as  shown  in  Fig-.  28.  A  large 
basin  is  filled  to  within  a  couple  of  centimetres  of  the  top  with 
water  (d).  Over  this  is  poured  carefully  a  layer  of  oil  2  cm. 
deep  (c),  upon  which  are  placed  one  or  several  small  floats  (/) 
with  wicks,  such  as  are  used  on  the  continent  for  night-lamps. 
Such  a  floating  burner  gives  an  especially  constant  heat.  As 
the  flame  cannot  be  chang-ed  in  size,  it  is  necessary  to  find 
the  right  distance  between  it  and  the  thermostat;  and  since 
this  must  be  kept  as  nearly  constant  as  possible  throughout 
the  day,  it  is  usual  to  employ  a  large  bowl  and  a  thin  layer  of 
oil,  so  that  the  wick  shall  not  recede  too  far  from  the  bottom 


54 


Bacteriological   Technology* 


of  the  brood-oven  as  the  oil  is  used  up.     The  small  wicks  are 
renewed  morning-  and  evening-. 

In  Figure  28  is  shown  a  still  cheaper  thermostat  than  that 
represented  in  Fig.  24.  It  has  no  regulator,  no  tube  for  show- 
ing the  height  of  water,  no  faucet  for  removing  this,  and  no 
attached  covering-  of  felt.  But  it  can  at  any  time  be  easily 


FIG.  28.— Simple  Brood -oven  Heated  by  an  Oil-lamp 

covered  by  rectangular  pieces  of  felt  or  flat-  sheets  of  cotton- 
batting,  of  which  four  can.be  sewed  together  at  their  edges, 
while  the  fifth,  over  the  door,  is  fastened  only  by  its  top  to  the 
upper  piece,  so  that  it  can  be  lifted  when  the  door  is  to  be 
opened,  while  it  is  tied  to  the  side  pieces  when  the  door  is 
closed. 


OHAPTEE  VII. 

BACTERIOLOGICAL  ANALYSIS  OF  FLUID,  SOLID,  AND 
GASEOUS  SUBSTANCES;  ESPECIALLY  OF  WATER,  THE 
SOIL,  AND  AIR. 

MICROSCOPIC  investigation  shows  very  incompletely  what 
living-  germs  are  present  in  a  preparation.  Different  appear- 
ing micro-organisms  may  represent  different  stages  in  the  de- 
velopment of  a  single  species,  and  bacteria  which  look  quite 
identical  may  belong  to  entirely  different  species.  Moreover, 
all  germs  of  bacteria  are  by  no  means  recognizable  as  such 
under  the  microscope;  and  the  living  are  not  certainly  distin- 
guishable from  the  dead.  If  the  question  is  merely  whether 
some  specified  virulent  form  occurs  in  a  sample  of  earth  or 
water,  in  case  this  form  is  pathogenic  for  animals,  the  most 
direct  answer  will  be  obtained  by  inoculation.  Water  is  sub- 
cutaneously  injected  into  a  suitable  animal.  Earth  is  placed 
in  a  little  pocket  made  in  the  subcutaneous  tissue.  Bacteria 
from  the  air  are  first  collected  in  a  sterilized  filter  of  sand  (cf. 
analysis  of  air),  which  is  then  introduced  into  such  a  pocket. 
But  a  satisfactory  bacteriological  analysis  cannot  to-day  be 
made  without  recourse  to  cultures.  It  is  of  especial  interest 
for  the  physician  to  know  these  methods,  since  they  afford 
the  most  important  introduction  to  hygienic  investigation  of 
water,  earth,  and  air.  Since  the  discovery  of  the  cholera 
spirillum  they  have  also  been  included  among  the  best 
methods  of  clinical  investigation.  An  exhaustive  bacteriolog- 
ical investigation  includes  a  determination  of  the  number  of 
bacteria  present,  as  well  as  their  identification;  but  for  easily 
understood  reasons,  in  both  hygienic  and  clinical  investiga- 
tions, qualitative  analysis  is  most  frequently  of  entirely 
greater  importance  than  quantitative. 


5  6  Bacteriological    Technology. 

A.  LIQUIDS. 
(With  especial  reference  to  water  analysis.) 

All  of  the  methods  indicated  in  Chapter  IV.  may  find  ap- 
plication in  the  bacteriological  analysis  of  fluids.  Of  these, 
we  usually  resort  to  dilution,  and  isolating-  the  g-erms  in  nutri- 
ent gelatin,  as  the  two  principal  methods. 

The  latter,  first  indicated  by  Koch,  and  developed  by  him 
to  a  hig-h  degree  of  completeness,  is  alone  sufficient  in  by  far 
the  greater  number  of  cases,  and  consequently  will  be  treated 
here  in  detail.  Koch's  method  consists  in  mingling  the  fluid 
containing-  germs  with  liquefied  gelatin,  which  is  then  spread 
in  a  thin  layer  and  allowed  to  solidify,  being-  protected  from 
atmospheric  contamination.  The  mixing-  and  spreading  can 
obviously  be  effected  in  a  great  variety  of  receptacles,  among 
which  are  four  deserving-  especial  attention. 

a.  Conical  Flasks. — A  medium-sized  flask  (Fig.  9,  VII.) 
containing-  a  thin  layer  of  g-elatin  in  the  bottom  is  warmed  just 
enough  to  melt  the  gelatin  (over-heating  may  destroy  the 
bacterian  g-erms).     By  aid   of  a  needle  or  pipette,  a  small 
quantity  of  the  liquid  to  be  analyzed  is  then  added,  with  the 
greatest  possible  rapidity  and  care.     The  two  are  carefully 
mingled,  but  without  shaking,  which  might  cause  the  forma- 
tion of  bubbles.     To  avoid  this,  the  flask  is  held  upright,  and 
several  times  carried  around  a  large  circle  in  a  horizontal 
plane.     The  fluid  is  set  in  so  active  movement  by  centrifugal 
force  that  the  commingling  is  perfect,  without  the  introduc- 
tion of  bubbles  into  the  gelatin,  which  is  then  allowed  to  solid- 
ify.    This  is  one  of  the  simplest  and  surest  methods  by  which 
a    Koch   isolation-culture  can  be  made.     One    disadvantage 
attends  it,  viz. :  the  isolated  colonies  are  not  accessible,  in  the 
flask,  for  microscopic  investigation,  and  less  accessible  for  ex- 
amination by  the  naked  eye  or  with  a  lens  than  in  the  follow- 
ing cases. 

b.  Test-tubes. — The  mixture  can  also  be  effected  in  a  test- 
tube,  on  the  inside  of  which  the  gelatin  is  then  allowed  to 
harden  in  a  thin  layer  (von  Esmarch).     For  such  "roll-cul- 
tures," wide  tubes  are  best,  with  about  10  cc.  of  nutrient  gela- 
tin.   Naturally,  one  may  add  the  fluid  as  indicated  in  Chap- 


Bacteriological    Technology.  57 

ter  V.,  and  subsequently  melt  the.  gelatin  over  a  water  bath, 
or  this  may  be  done  first.  The  mingling  of  the  two  is  effected 
as  in  a.  The  distribution  of  the  gelatin  over  the  wall  of  the 
tube  is  secured  simultaneously  with  its  hardening  in  the  fol- 
lowing manner:  The  projecting  portion  of  the  cotton  plug  is 
clipped  or  singed  off,  and  the  whole  covered  with  a  tight-fitting 
rubber  cap  to  keep  the  cotton  dry,  and  the  tube  laid  in  a  dish 
of  cold  water,  preferably  ice-water.  While  the  tube  floats  on 
the  water,  it  is  kept  with  one  hand  as  nearly  horizontal  as 
possible,  while  with  the  other  it  is  rotated  until  the  gelatin 
has  become  solid  in  a  uniform  layer  over  the  entire  inside  of 
the  tube,  which  is  then  dried  off  and  the  rubber  cap  removed. 
Occasionally  the  cotton  plug  becomes  covered  with  so  thick  a 
layer  of  gelatin  as  to  prevent  the  access  of  air  to  the  culture 
in  an  injurious  manner.  In  this  case,  cotton  and  gelatin  may 
both  be  pierced  by  a  sterile  platinum  needle.  The  advantage 
of  the  test-tube  over  a  flask  is  that  it  takes  up  less  room,  and 
its  contents  are  somewhat  more  readily  accessible  for  investi- 
gation. 

c.  Q-lass  Trays. — It  is  convenient  to  use  a  pair  of  shallow 
trays  (Fig.  12)  about  20  cm.  in  diameter  and  1.5  cm.  deep,  which 
are,  of  course,  first  to  be  sterilized,  well  wrapped  in  paper, 
from  which  they  are  removed  only  immediately  before  being 
used.  Mingling  the  fluid  which  contains  bacteria  and  the 
gelatin,  is  effected  in  a  test-tube  as  in  the  last  case.  The  cot- 
ton plug  is  removed  and  the  mouth  of  the  tube  flamed  [it  is 
better  to  clip  the  cotton  off  close  to  the  edge  of  the  tube,  push 
the  plug  down  a  little  way,  and  then  flame  until  the  cotton 
browns  a  little.  After  cooling,  the  plug  is  easily  removed 
with  sterile  forceps,  and  the  gelatin  poured. — W.  T.],  and  as 
soon  as  it  has  cooled  the  contents  are  poured  as  rapidly  as 
possible  into  the  lower  (smaller)  tray,  while  the  other,  serving 
as  a  cover,  is  pushed  a  little  to  one  side.  Since  the  tube  is 
never  entirely  emptied,  but  a  small  quantity  of  gelatin  re- 
mains in  it,  it  is  best  to  label  and  preserve  it  as  complemen- 
tary to  the  tray  culture. 

A  uniform  layer  of  gelatin  is  seldom  secured  in  these  trays. 
The  bottom  is  not  plane,  but  as  a  rule  a  little  elevated  in  the 
middle.  But  the  consequent  difference  in  thickness  of  differ- 
ent parts  of  the  gelatin  has  its  advantage,  for  when  many 
germs  are  present  in  the  fluid  to  be  analyzed,  and  they  are 


5  8  Bacteriological   Technology. 

too  closely  crowded  in  the  thick  peripheral  part  of  the  layer, 
they  are  more  isolated  in  the  central  thin  portion. 

The  colonies  in  such  tray-cultures  are  much  more  easily 
accessible  for  examination  with  the  naked  eye  or  lens  than  in 
a  or  b.  Examination  is  frequently  rendered  somewhat  difficult 
because  the  under  side  of  the  cover  becomes  clouded.  In  this 
case  it  may  be  replaced  by  a  dry  and  sterile  glass  plate  during 
the  necessary  inspection.  [Non-liquefying  species  are  readily 
examined  by  inverting  the  trays.]  These  cultures  are  also 
far  better  for  microscopic  examination  than  those  in  flasks  or 
test-tubes,  especially  if  smaller  trays  (5  or  6  cm.  in  diameter) 
are  employed;  but  they  are  far  from  being  as  good  in  this  re- 
spect as  plate-cultures. 

d.  Glass  Plates. — This  method,  the  "plate  method"  of 
Koch,  of  which  the  three  preceding  are  only  modifications,  dis- 


a  b 

FIG.  29.— Slide  Coated  with  Gelatin  Crossed  by  Fourteen  Inoculation  Scratches  bearing 
many  Colonies.    At  6,  a  mould  colony  due  to  accidental  contamination  from  the  air. 

tinguishes  itself  from  these  not  only  by  a  certain  elegance, 
but  especially  in  allowing  a  microscopic  examination  of  the 
colonies  anywhere  on  the  gelatin,  with  high  magnification. 
Koch's  first  plate-cultures  were  undertaken  in  the  following 
manner:  The  gelatin  was  spread  in  an  elongated  drop  on  a 
common  microscope  slide,  by  the  use  of  a  pipette.  When  it 
had  become  solid,  it  was  inoculated  by  a  bent  platinum  needle 
(Fig.  21,  d)  dipped  in  the  fluid  containing  germs,  and  then 
used  to  make  a  series  of  transverse  scratches  on  the  gelatin 
(Fig.  29).  In  the  course  of  a  few  days  colonies  of  the  bacteria 
appear  in  the  inoculation  furrows, — closest  and  often  entirely 
coalescent  in  those  made  first  (a),  fewer  and  well  separated  in 
the  last.  Assistance  in  recognizing  contamination  of  such 
scratch-cultures  from  the  air,  is  afforded  by  the  fact  that  they 
almost  always  appear  between  the  scratches,  as  is  the  case 
with  the  mould  colony  &,  in  Fig.  29.  In  two  respects  the 


Bacteriological    Technology.  59 

plate  method  is  inferior  to  the  three  preceding.  From  the 
nature  of  the  inoculation,  the  plates  are  more  exposed  to  at- 
mospheric germs;  and  the  distribution  of  the  gelatin  over  the 
plates  takes  more  time  and  care,  since  it  must  be  kept  from 
running  over  the  edges,  which  is  best  avoided  by  the  use  of 
suitable  cooling  appliances  which  cause  more  rapid  solidifica- 
tion. 

The  apparatus  needed,  and  the  method  of  starting  a  plate 
culture  are,  in  detail,  the  following: 

Rectangular  glass  plates  6.5  x  12  cm.  are  washed,  polished 
with  chamois  skin  and  sterilized  at  150°  C.,  well  wrapped  in 
paper  from  which  they  are  first  removed  immediately  before 
use.  [For  convenience,  the  sheet-iron  boxes  used  by  the  Ger- 
mans to  hold  sterilized  plates,  are  to  be  recommended. — W.  T.] 

The  fluid  containing  bacteria  is  mingled  with  the  gelatin 
as  explained  above  (p.  56),  in  a  cotton-plugged  test-tube  con- 


FIG.  30. — A  Simple  Cooling  Apparatus  for  Plate-cultures. 

taining  about  5  cc.  of  peptonized  gelatin,  a  quantity  adapted 
to  the  size  of  plate  recommended ;  especial  care  being  taken 
to  warm  the  gelatin  only  a  little  above  the  melting  point. 

A  serviceable  cooling  apparatus  is  easity  made  as  show^n 
in  Fig.  30.  On  an  ordinary  pie-plate,  (a)  is  set  a  preparation 
glass  (b)  with  ground  rim,  several  pieces  of  ice  are  placed  in 
this  and  water  is  carefully  poured  in  until  the  vessel  is  full, 
with  a  slightly  convex  surface,  but  not  running  over.  A  well- 
cleaned  square  plate  of  glass  (d)  is  now  let  down  on  the  sur- 
face of  the  water  so  as  to  wet  it  everywhere  and  thus  avoid 
air-bubbles,  a  little  water  running  over  into  the  plate  as  this 
is  done.  Finally,  a  sterilized  glass  dish  (e)  is  inverted  on  the 
plate  to  serve  as  a  bell-glass. 

One  of  the  glass  plates  is  unpacked  and  quickly  laid  under 
the  bell-glass  (Fig.  30,  /),  where  it  is  rapidly  cooled  by  the  ice 
water.  The  plug  is  removed  from  the  infected  tube,  the  mar- 
gin of  which  is  quickly  flamed  [here,  as  above,  p.  47,  it  is 


60  Bacteriolog  ical    Technology. 

preferable  to  flame  the  tube  before  removing1  the  cotton  plug-. 
— W.  T.],  after  which  the  bell-glass  is  raised,  and  the  still  fluid 
g:elatin  carefully  poured  over  the  glass  plate,  care  being  taken 
not  to  let  it  flow  too  near  the  margin.  The  bell-glass  is  re- 
placed to  protect  the  plate  while  the  gelatin  solidifies.  The 
more  exactly  horizontal  the  plate  stands,  the  better.  If  the 
gelatin  runs  in  a  given  direction,  because  the  plate  is  not  level, 
it  is  possible  to  carefully  lift  the  apparatus  with  both  hands 
and  check  this  by  adjusting  its  position,  until  the  gelatin  is 
sufficiently  hardened.  It  is  naturally  more  convenient  to  level 
the  plate  beforehand  by  using  a  tripod  and  level  (Fig.  31).  If 
it  solidifies  too  quickly,  before  spreading  over  a  sufficiently 
large  part  of  the  giass,  it  may  be  spread  by  use  of  a  sterile 
glass  rod,  warmed,  but  not  too  hot.  The  test-tube,  in  which 


FIG.  31.— Leveling  Tripod  with  Round  FIG.  32.— Bench  of  Sheet  Zinc  for  Support- 

Level  (6)  and  Plate.  ing  a  Plate-culture. 

a  few  drops  of  gelatin  always  remain,  is  again  plugged  with 
the  original  cotton;  labelled,  rotated  for  several  minutes  and 
laid  horizontally,  so  that  this  vestige  solidifies  in  a  thin  layer, 
and  kept  as  supplementary  to  the  plate-culture,  which,  as 
soon  as  it  has  hardened,  is  labelled  and  quickly  removed  to  the 
moist  chamber. 

For  a  single  plate,  a  pair  of  shallow  glass  trays  (Fig.  12) 
may  be  used  as  a  moist  chamber.  But  as  a  rule  it  is  neces- 
sary to  make  several  plate-cultures  of  the  same  fluid.  In  this 
case  a  pair  of  larger  deep  trays  are  used,  or  a  chamber  may 
be  improvised.  Before  use,  it  is  to  be  rinsed  with  0.1  per  cent 
sublimate  solution,  and  a  couple  of  sheets  of  filter  paper,  well 
wet  with  the  same  solution,  are  laid  in  the  bottom.  The 
plates  are  set  in  such  a  moist  chamber,  on  small  glass  or 
metal  benches  which  can  be  set  over  one  another.  Sheet-zinc 
benches  of  the  form  shown  in  Fig-.  32  are  very  suitable  because 
of  their  durability.  They  can  easily  be  sterilized  at  150°  C. 
before  use. 

The  examination  of  the  plates  by  the  naked  eye  is  usually 


Bacteriological    Technology.  61 

made  over  either  a  white  or  black  background  (sheets  of  paper 
or  tiles),  as  many  differences  in  color  between  the  colonies  are 
only  seen  well  in  this  way.  It  is  also  to  be  remembered  that 
colonies  of  the  same  species  and  of  equal  age  may  present 
great  differences  in  size,  form,  and  color,  according-  as  they 
grow  at  the  surface  or  immersed  in  the  gelatin.  Counting-  the 
colonies  is  best  effected  by  the  use  of  a  lens  of  low  power,  and 
is  much  easier  if  the  plate  is  laid  on  another  divided  into 
square  centimetres.  [When  it  can  be  afforded,  one  of  the  sim- 
ple counting-  appliances  used  by  the  Germans  in  water-analy- 
sis, is  to  be  strongiy  recommended. — W.  T.] 

The  microscopic  examination  of  the  plate  is  not  difficult, 
even  with  rather  high  powers.  To  be  able  to  examine  every 
part  of  the  plate,  a  smaller  size  than  the  one  recommended 
may  be  necessary,  if  the  stage  of  the  microscope  is  not  quite% 
larg-e.  Since  it  is  impossible  to  calculate  beforehand  the 
number  of  germs  added  to  the  gelatin,  in  each  case  it  is  cus- 
tomary to  prepare  three  plate-cultures  at  a  time  of  three  de- 
grees of  dilution.  This  is  best  done  as  follows :  The  gelatin 
is  liquefied  in  three  test-tubes,  which  are  numbered  1,  2,  and  3. 
After  1  has  been  infected  by  use  of  needle  or  pipette,  and  the 
added  material  well  distributed,  2  is  infected  from  it ;  and,  in 
the  same  way,  3  from  this.  The  gelatin  is  poured  on  the 
plates  as  above.  In  this  way,  it  is  highly  probable  that  one 
of  the  three  plates  will  contain  a  suitable  number  of  colonies, 
whether  these  are  wanted  so  numerous  that  the  culture  can 
be  used  for  a  tolerably  reliable  estimate  of  the  number  of 
germs  present  in  the  fluid  (c/.  water  analysis,  p.  64),  or  so 
few  that  a  nearly  absolute  guarantee  is  given  of  the  purity  of 
each  colony,  and  transfers  may  be  made  from  them  without 
difficulty. . 

It  should  be  remembered,  as  advised  above,  to  save  the 
three  test-tubes  after  distributing  the  gelatin  in  a  thin  layer 
by  rolling  them  several  times.  Where  the  object  is  to  get  an 
exact  determination  of  the  number  of  germs  in  the  original 
fluid,  it  is  necessary  to  include  those  remaining  in  the  tube, 
and,  independently  of  this,  it  is  always  an  advantage  to  have 
two  isolation-cultures  of  each  dilution. 

Soyka  obtains  a  large  number  of  very  small  plate  cultures 
by  using  pairs  of  trays  which  differ  from  those  shown  in  Fig. 
12  in  being  made  of  glass  1.5  mm.  thick,  with  a  bottom  50 


62  Bacteriological    Technology. 

square  cm.  in  area,  and  walls  only  3  to  5  mm.  Seven  round 
hollows  are  ground  in  the  lower  tray,  like  those  in  hollow- 
ground  slides  (Fig-.  57).  Before  being  used,  they  are  slightly 
warmed,  gelatin  is  placed  in  each  of  these  depressions  by  using 
a  pipette,  one  of  the  drops  is  quickly  inoculated  with  a  looped 
platinum  needle,  carefully  stirred,  and  a  transfer  made  from 
this  to  the  second,  etc.,  the  upper  tray  or  lid  being  put  in  place 
after  all  are  inoculated.  If  the  pipette  is  accurately  gradu- 
ated, and  the  quantity  of  fluid  carried  b}r  the  platinum  loop 
previously  determined  by  weighing  it  dry  and  again  with  its 
drop  of  water,  it  is  claimed  by  Soyka  that  a  sufficiently  relia- 
ble determination  .may  be  made  of  the  number  of  germs.  This 
modification  of  the  customary  Koch  method  of  plating-  out 
cultures  appears  to  me  excellent  in  its  plan,  but  the  form  of 
,the  trays  and  the  limited  size  of  the  hollows  do  not  seem  so 
good.  For  microscopic  examination,  I  would  recommend 
rectangular  glass  trays  (e.  g.,  12x5  cm.),  the  lower  one  divided 
by  elevated  ridges  into  eight  quadrangular  spaces,  so  that  the 
surface  of  the  several  cultures  should  be  larger. 

In  the  preceding  descriptions  we  have  always  assumed 
that  the  isolation  was  to  be  effected  in  either  gelatin  or  agar. 

1.  Gelatin. — This  is  most  easily  used,  since  it  may  be  melted 
and  kept  fluid  at  a  relatively  low  temperature;  yet  the  fact 
that  many  bacteria  and  moulds  liquefy  (peptonize)  the  gelatin 
is  a  disadvantage  that  often  becomes  very  evident  in  such 
analyses. 

2.  Agar- Agar. — Most  bacteria  cause  no  liquefaction  of  this 
substance,  which  is  nevertheless  not  so  good  as  gelatin  for 
plate-cultures,  because  of  the  difficulty  of  melting  it  and  its 
somewhat  slighter   transparency,   and   its  tendency  not  to 
adhere  to  the  glass;  still,  it  may  be  used  well  for  analyses  of 
this  character  if  the  following  precautions  are  taken.    When 
the  ag-ar  has  been  melted  in  the  test-tubes,  which  does  not 
occur  below  90°  C.,  these  are  set  aside  to  cool  for  a  few  mo- 
ments and,  before  their  contents  begin  to  solidify,  placed  in  a 
water-bath  kept  at  40°  0.,  at  which  point  the  agar  remains 
fluid,  while  the  liquid  containing  bacteria  can  safely  be  added, 
without  danger  of  killing  any  of  the  germs. 

While  it  is  usually  necessary  when  gelatin  is  used  to  hasten 
its  solidification  by  cold  (or  iced)  water,  this  must  commonly 
l>e  retarded  when  agar  is  employed,  by  placing  the  plates 


Bacteriological    Technology.  63 

over,  or  rolling1  the  tubes  in,  lukewarm  water,  to  prevent  the 
agar  from  forming1  lumps  and  as  a  result  hardening  with  an 
uneven  surface.  Since  ag-ar  adheres  so  poorly  to  glass  that  it 
occasionally  entirely  separates  from  the  plate  and  slips  off, 
Fraenkel  advises  the  use  of  a  drop  of  sealing-  wax  on  each  cor- 
ner of  the  plate. '  [Where  ag-ar  must  be  used  in  plating1  out 
bacteria,  it  is  much  better  to  do  this  in  trays  (supra,  c),  by 
which  the  curling  of  the  ag-ar  is  avoided. — W.  T.] 

3.  Agar-Gelatin,  prepared  after  Jensen's  formula  (supra, 
pp.   25,  26)  combines  some  of  the  g-ood  qualities  of  "both  g-ela- 
tin  and  agar.     It  is  very  conveniently  used  for  plate-cultures, 
and,  as  a  rule,  is  to  be  recommended  in  preference  to  either. 
The  surest  way  of  keeping1  it  fluid  during1  the  process  of  inoc- 
ulation, is  to  set  the  test-tubes  in  a  water  bath  at  30°  to  40°  C. 

4.  Serum  cannot  be  used  in  this  manner,  since  it  is  coagu- 
lated only  by  prolonged  heating1  to  a  degree  likely  to  prove 
fatal  to  the  g-erms.     If  it  must  be  employed,  one  of  two 
methods  must  be  adopted:   Scratch-cultures  may  be  succes- 
sively made  with  an  infected  bent  platinum  needle,  in  a  larg-e 
number  of  test-tubes,  the  colonies  being-  so  few  in  the  last  of 
the  series  as  to  be  separate  (c/.  the  original  scratch-cultures 
on  slides,  p.  58) ;  or  the  fluid  to  be  used  may  be  diluted  with 
sterile  water,  and  a  small  drop  of  the  mixture  allowed  to  flow 
about  over  the  surface  of  the  serum,  in  the  hope  that  the 
germs  will  settle  on  it  at  a  distance  from  one  another.      But 
it  should  be  borne  in  mind  that  this  is  an  exceptional  method 
which  does  not  give  nearly  so  complete  a  separation  of  the 
g-erms  as  the  careful  distribution  in  melted  gelatin. 

Water  Analysis. — The  bacteriological  analysis  of  drinking- 
water  is  undertaken  according-  to  the  rules  given  above.  The 
following-  precautions  are  also  to  be  taken :  The  water  is  col- 
lected in  sterilized  glass  flasks,  with  cotton  or  glass  stoppers. 
If  it  is  taken  from,  a  faucet  or  hydrant,  it  is  allowed  to  run 
for  some  minutes  before  any  is  g-athered,  and  care  must  be 
taken  to  prevent  the  contamination  of  the  stopper  from  any 
source  (by  placing-  it  in  a  sterile  glass  box,  Figs.  11  and  13, 
etc.).  Samples  are  taken  from  accessible  bodies  of  water  in 
sterilized  pipettes,  and  quickly  distributed  in  sterile  flasks. 
In  case  of  deeper,  less  accessible  supplies,  a  weighted  sterilized 
flask  is  lowered  and  drawn  up  when  full,  its  contents  being-  at 
once  divided  between  several  smaller  bottles.  The  analysis 


64  Bacteriological    Technology. 

must  be  made  as  soon  as  possible  after  the  sample  is  taken; 
even  a  few  hours'  delay  at  ordinary  room  temperature  effects 
a  great  increase  in  the  number  of  germs.  If  it  is  necessary  to 
send  samples  of  water  to  any  considerable  distance,  or  to  let 
them  stand  for  some  time  before  they  can  be  analyzed,  they 
must  be  packed  in  ice. 

The  quantity  of  water  to  be  added  to  each  tube  of  gelatin, 
depends  upon  the  number  of  germs  present  in  it.  When  few 
are  present,  an  entire  cubic  centimetre  may  be  used.  Occa- 
sionally -gV  cc-  suffices.  Water  that  contains  germs  in  especial 
abundance  must  be  diluted  with  occasionally  several  thousand 
times  its  bulk  of  sterilized  water,  and  a  drop  may  even  then 
give  a  large  number  of  colonies  on  the  gelatin  plate,  Botton 
puts  10  colonies  to  the  plate  as  a  minimum,  and  5,000  as  a 
maximum,  if  good  results  are  to  be  secured.  With  fewer  than 
10,  accidental  contaminations  easily  vitiate  the  results.  If 
the  plate  contains  too  many,  the  task  of  counting  them  is 
more  difficult,  and  some  of  them  may  not  develop  enough  to 
become  visible.  It  is  best  to  keep  the  plates  at  20°  to  22° 
C.,  and  to  count  the  colonies  on  the  third  or  fourth  day. 

B.  SOLID  SUBSTANCES. 

The  bacteriological  analysis  of  solids  is  quickly  described 
after  the  detailed  account  of  fluid  analysis.  It  may  be  carried 
out  exactly  like  the  latter,  by  first  quickly  dropping  the  sub- 
stance into  the  melted  gelatin,  in  which  the  germs  are  washed 
out  and  distributed  as  far  as  possible  in  the  manner  indicated 
for  the  analysis  of  water.  If  it  is  not  certain  that  the  germs 
can  be  sufficiently  washed  out  even  by  continued  agitation  in 
the  liquefied  gelatin,  this  may  be  effected  by  violent  and  pro- 
longed shaking-  in  sterilized  water  or  bouillon,  which  is  after- 
ward investigated  according  to  the  rules  given  for  fluids.  It 
is  also  possible,  after  such  treatment,  to  add  an  equal  quan- 
tity of  melted  20-per-cent  nutrient  gelatin  to  the  water,  and, 
after  careful  mixing,  to  spread  this  out  in  one  of  the  described 
methods. 

Fraenkel  recommends  the  first  method  for  soil  analyses, 
as  the  result  of  a  large  number  of  comparative  experiment.4 
Because  of  the  great  difference  between  different  samples  of 
soil,  as  regards  the  amount  of  water  present,  he  further  ad- 


Bacteriological    TecJmology.  65 

vises  measuring-  the  samples  in  preference  to  weighing1  them, 
using-  for  this  purpose  a  sharp-edged  spoon  holding  about  -^ 
cc.  A  level  spoonful  of  dirt  is  poured  into  a  test-tube  contain- 
ing melted  gelatin,  and  broken  up  and  distributed  in  this  as 
thoroughly  as  possible  by  the  use  of  a  stout  platinum  needle, 
and  by  agitating  the  glass,  after  which  the  procedure  is  the 
same  as  for  analysis  of  fluid.  The  same  writer  advises  the 
use  of  von  Esmarch's  tube-method  (supra,  p.  56),  and  counts 
the  colonies  two  days  after  preparing  the  tubes.  But  the 
common  plate -method  is  equally  applicable  and  occasionally 
even  preferable,  especially  when  the  samples  contain  many 
liquefying  forms,  as  is  generally  the  case  with  the  surface  soil. 
A  great  deal  of  the  difficulty  from  this  cause  may  be  avoided 
by  the  use  of  agar-gelatin.  On  the  other  hand,  the  tube- 
cultures  admit  of  prolonged  observation,  and  in  this  way  of 
the  detection  of  those  very  slowly  developing  forms  which 
frequently  occur  in  earth  (c/.  Chapter  XII.,  on  disinfection). 
Samples  from  the  deeper  soil,  which,  as  the  studies  of  Koch 
and  Fraenkel  show,  always  contain  very  few  germs,  can  only 
be  obtained  with  the  certain  exclusion  of  dirt  from  above  by 
the  use  of  a  special  boring  apparatus  (made  by  Muencke,  of 
Berlin).  Like  water,  soil  must  be  investigated  very  soon  after 
collection,  since,  especially  in  samples  from  some  depth,  there 
is  a  very  rapid  multiplication  of  the  bacteria,  which  may  be- 
come a  thousand-fold  more  numerous  than  at  first,  in  the 
course  of  a  few  days  (Fraenkel).  The  reason  for  this  increase, 
which  cannot  be  prevented  by  the  use  of  ice,  is  still  unknown. 
In  his  first  soil  analyses,  Koch  used  the  simple  method  of 
sprinkling  the  earth  over  the  surface  of  gelatin.  Naturally, 
no  certain  separation  of  the  germs  is  possible  by  this  means, 
but  a  good  notion  is  obtained  of  the  kinds  of  bacteria  and 
moulds  present  in  the  sample.  It  is  best  for  this  purpose  to 
pour  the  sterile  nutrient  gelatin  upon  plates  or  in  trays, 
where  it  is  allowed  to  harden.  The  earth  is  collected  with 
the  usual  precautions  in  sterile  test-tubes  or  flasks  plugged 
with  cotton.  Immediately  before  use,  the  cotton  is  removed 
and  a  layer  of  filter  paper  quickly  tied  over  the  mouth  of  the 
glass.  Holes  are  made  in  the  paper  with  a  flamed  pin,  and 
through  these  the  dirt  is  dredged  upon  the  gelatin,  so  that 
the  fine  particles  do  not  lie  too  close  together. 


66 


Bacteriological    TecJinology. 


a 


C.  Am. 

The  micro-organisms  which  float  in  the  atmosphere,  and 
compose  an  important  part  of  its  so-called  dust,  have  been  in- 
vestigated for  a  long-  time  in  many  different  ways,  and  with 
more  zeal  than  those  of  water  and  the  soil;  on  the  one  hand  with 
reference  to  the  theory  of  spontaneous  generation,  and  on  the 
other,  because  too  much  weight  was  laid  upon  the  importance 
of  the  air  as  a  carrier  of  nearly  all  contagious  matters.  'But 
the  time  and  energy  be- 
stowed are  sadly  out  of  pro- 
portion to  the  small  result- 
ant gain  in  knowledge  of  the 
life  histor3r  and  mode  of 
transportation  of  pathogenic 
"bacteria. 

Two  modes  of  collecting 
air  and  its  organisms  are  in 
use: — the  dust  is  allowed  to 
settle  by  its  own  weight,  or 
it  is  drawn  into  a  suitable 
apparatus  by  using  an  aspi- 
rator. 

Aspirators.  —  A  simple 
aspirator,  easy  of  transpor- 
tation, is  that  shown  in  Fig.  ' 

'  FIG.  33.— Aspirator  Made  from  Two  Flasks. 

33.    Two  large  conical  flasks 

(I.  and  II.)  are  furnished  with  rubber  stoppers  and  glass  tubes 
arranged  (Fig.  33)  as  for  wash  bottles,  and  connected  with  a  rub- 
ber tube  in  the  manner  shown.  A  pinch-cock  is  placed  at  c,  and 
the  upper  flask  filled  with  water.  When  the  cock  is  opened  and 
suction  applied  at  a  (or  the  upper  flask  tipped)  till  the  siphon  is 
filled,  the  water  will  flow  without  interruption  from  I.  into  the 
empty  flask  II.,  inducing  a  uniform  and  continuous  suction  of  air 
into  b,  until  I.  is  empty.  By  pouring  the  water  back  into  I.  (or 
reversing  the  relative  position  of  the  flasks)  the  suction  can  be 
recommenced.  Knowing  the  volume  of  water  used,  and  the 
time  taken  to  empty  one  flask  into  the  other,  the  amount  of 
air  sucked  into  the  apparatus  during  a  given  time  is  also 
known.  The  rapidity  with  which  the  water  flows  from  flask 


Bacteriological    Technology. 


67 


to  flask  can  by  regulated  by  the  pinch-cock,  or  by  the  use  of 
glass  tubes  of  various  calibre,  inserted,  somewhere  in  the 
rubber  tube.  A  number  of  such  pieces  of 
tubing-  are  made  ready,  and  the  time  re- 
quired for  each  to  permit  the  passage  of 
a  liter  of  water  is  experimentally  deter- 
mined and  marked  on  it  with  a  writing  dia- 
mond, so  that  according  to  circumstances  a 
larger  or  smaller  tube  is  employed  (Hesse).  • 
Another  form  of  aspirator  is  that  shown 
above  in  Figure  7. 

c.  A  drip-aspirator  is  shown  in  Figure 
34  in  which  the  constant  stream  from  a 
faucet  can  be  used  as  a  means  of  suction. 
As  the  water  falls  in  small  portions  from 
the  faucet,  through  the  tubes  a  and  &, 
the  little  columns  of  fluid  carry  along  be- 
tween them  larger  and  smaller  columns 
of  air,  producing  at  c  a  powerful  suction, 
which,  however,  is  not  so  uniform  as  in  the 
other  two  aspirators.  [The  German  deal- 
ers supply  a  very  simple  aspirator  essen- 
tially on  this  model,  consisting  of  a  T- 
shaped  brass  tube  representing  abc,  joined 
by  suitable  corks  and  rubber  tubing  with 
a  flexible  lead  tube  some  yards  long,  which, 
being  non-collapsible,  makes  a  very  good 
connection  with  the  other  apparatus  em- 
ployed.— W.  T.]  Nor  does  the  apparatus 
directly  indicate  the  quantity  of  air  sucked 
through,  like  the  others,  so  that  if  this  is 
to  be  determined  it  must  be  used  in  connec- 
tion with  a  gas-meter. 

I.  Collection  of  Dust  for  Direct  Mi- 
croscopical Examination.  —  The  dust 
which  has  accidentally  accumulated  in  dif- 
ferent places,  often  in  a  thick  layer,  can  be 
simply  collected,  even  if  the  layer  is  a 
slight  one,  or  is  easily  swept  on  to  a  piece 
of  paper  with  a  feather.  Small  quantities  of  the  dust  col- 
lected in  this  manner  are  mingled  with  a  drop  of  glycerin  or 


FIG.  34.— Drip  Aspirator 
(Filter  Pump). 


68 


Bacteriological    Technology. 


other  fluid  on  a  slide  and  subjected  to  microscopic  and  micro- 
chemical  examination  in  the  usual  manner. 

If  the  dust  is  to  be  collected  for  a  given  time  and  place, 
this  is  best  done  by  the  use  of  a  so-called  aeroscope  (Ponchet, 
1859).  Of  the  various  models,  only  Schnauer's  will  be  de- 
scribed here — a  form  which  can  easily  be  put  together  in  any 
laboratory.  It  consists  (Fig-.  35)  of  a  bell-glass  (a)  with  ground 
base  and  short  neck.  A  bottle  with  the  bottom  broken  off 
and  the  lower  end  ground  flat  can  be  made  to  serve  the  same 
purpose.  The  neck  is  fitted  with  a  rubber  stopper  provided 
with  two  holes,  in  one  of  which  a  short  glass  tube  (d)  is  fitted, 
while  a  longer  tube  (e),  bent  in  an  arch  above  and  drawn  out 
to  a  point  with  an  opening  of  about  1  mm.  at  the  other  end, 
passes  through  the  second.  The 
ground  base  of  the  bell-glass  is 
smeared  with  vaseline  and  pressed  air 
tight  against  a  glass  plate  (b).  Un- 
der the  bell-glass  is  placed  a  slide  (c) 
on  which  is  a  small  drop  of  a  steril- 
ized solution  of  grape  sugar  1  -part, 
and  glycerin  2  parts  (Miquel).  The 
tube  e  is  then  adjusted  so  that  its 
point  is  about  1  mm.  above  the  surface 
of  the  drop,  and  the  tube  d  is  con- 
nected with  an  aspirator.  As  soon 

FIG.  35.— Schoenauer's  Aeroscope. 

as  the  suction  begins,  the  air,  and  the 

dust  suspended  in  it,  flow  through  the  curved  tube  directly 
against  the  drop  of  glycerin,  in  which  the  flow  of  air  causes 
a  slight  depression,  and  where  a  large  part  of  the  dust  is 
stopped.  A  part  settles  in  the  curved  tube,  which  must, 
nevertheless,  have  this  form  in  case  the  apparatus  is  to  be 
used  in  the  open  air  in  rainy  weather. 

When  the  aspirator  has  operated  long  enough,  the  slide  is 
removed,  and  the  dust  uniformly  distributed  through  the 
glycerin  with  a  sterilized  needle;  a  cover  glass  placed  on  it, 
and  it  is  ready  for  microscopic  and  micro-chemical  examina- 
tion. 

Preparations  of  this  sort  give  a  good  notion  of  many  of  the 
inorganic  and  organic  substances  which  float  in  the  air  in 
enormous  quantities,  e.g.,  coal  dust,  sand,  small  crystals  of 
various  salts,  woollen  and  cotton  threads,  hair,  starch-grains, 


Bacteriological    Technology.  69 

fragments  of  vegetable  tissue,  pollen,  etc.  .  Many  mould  spores 
are  also  recognizable,  but  the  germs  of  bacteria  are  too  small 
and  too  little  characteristic  to  be  identified  in  such  prepara- 
tions. The  number  and  character  of  such  germs  present  in 
the  air  at  a  given  time  can  only  be  determined  by  sowing  the 
dust  in  suitable  culture -vessels. 

II.  Collection  of  Dust  for  Cultures. —  Very  pretty  and 
useful  preparations  are  obtained  by  simply  letting  the  germs 
settle  from  the  air  upon  the  surface  of  gelatin.  Several  pairs 
of  shallow  glass  trays  (Fig.  12)  are  sterilized  at  150°,  each  pair 
wrapped  singly  in  paper.  The  nutrient  gelatin  (most  com- 
monly peptonized  meat  infusion  with  agar-gelatin)  is  carefully 
and  rapidly  poured  in  a  shallow  layer  into  the  smaller  tray, 
which  is  at  once  covered  with  the  other  (cf.  p.  63).  When 
the  gelatin  has  solidified,  the  trays  are  wrapped  up  and  taken 
to  the  place  where  they  are  to  be  used.  Here  they  are  un- 
packed, the  lids  (i.e.,  the  larger,  upper  trays)  taken  off,  and 
the  gelatin  is  left  exposed  to  the  air  for  a  longer  or  shorter 
time,  varying  with  the  abundance  of  germs  from  a  few  min- 
utes to  an  hour.  After  replacing  the  lids,  the  trays  are  set 
aside  at  room  temperature,  for  observation,  the  germs  begin- 
ning to  show  evidence  of  germination  after  a  few  days. 

It  is  best  to  use  the  sterilized  paper  to  wrap  the  lids  in 
while  the  gelatin  is  uncovered,  and,  after  they  are  replaced, 
to  again  wrap  the  pairs  of  trays  in  it  for  their  return  to  the 
laboratory.  Instead  of  pouring  the  gelatin  directly  into  trays, 
it  may,  .of  course,  be  spread  on  a  glass  plate,  which  is  then  set 
within  a  pair  of  trays. 

Simple  as  this  method  is,  it  gives,  as  has  been  said,  very 
useful  results.  This  may  easily  be  shown  by  exposing  a  num- 
ber of  trays  of  gelatin  simultaneously  and  for  the  same  length 
of  time  in  two  different  places.  The  differences  in  number  and 
kinds  of  atmospheric  germs  in  different  localities  is  then  very 
evident.  It  is  to  be  recommended  for  the  qualitative  air  anal- 
yses most  frequently  called  for,  but  it  does  not  give  the  num- 
ber of  germs  contained  in  a  given  quantity  of  air.  A  number 
of  methods  and  appliances  for  this  quantitative  analysis  of 
atmospheric  air  have  been  indicated,  but  there  is  at  present  a 
difference  of  opinion  as  to  which  is  to  be  preferred.  I  have 
not  sufficient  experience  in  this  directior.  to  pronounce  very 
authoritatively  on  the  question,  but  I  have  no  doubt  the  air- 


7°  Bacteriological    Technology. 

filters  using  (insoluble  or  soluble)  powders  are  to  be  given  the 
preference.  In  view  of  this  difference  of  opinion  it  must  be  a 
satisfaction  to  know  that  at  present  the  exact  determination 
of  the  number  of  germs  in  the  air  is  not  of  very  great  import- 
ance. I  shall,  therefore,  merely  describe  briefly  the  choice  of 
apparatus  representing  the  various  systems,  viz.,  automatic 
suction  balloons,  settling  apparatus,  such  in  which  the  air  is 
allowed  to  bubble  through  fluid  or  gelatinized  contents,  and 
filters,  which  may  be  insoluble  or  soluble. 

The  automatic  balloon  was  first  used  by  Pasteur  in  1860. 
As  shown  in  Fig.  36,  it  is  at  once  culture  vessel,  aspirator,  and 
gasometer.  It  consists  of  a  globular  flask  with  a  long  neck 
drawn  out  into  a  capillary  tube.  The  tube  is 
hermetically  sealed,  and  the  flask  sterilized  at  150° 
C.  When  cool,  the  point  is  broken  off,  the  air 
within  the  balloon  is  warmed  a  little  over  a  burner, 
and  the  tip  dipped  into  the  sterilized  culture-fluid. 
As  it  cools,  a  few  drops  of  fluid  pass  into  the  flask, 
this  is  carefully  heated  to  the  boiling  point  over 
the  burner,  and,  while  the  steam  is  escaping  from 
the  opening,  this  is  again  clipped  into  the  fluid,  a 
greater  quantity  of  which  is  sucked  in.  When  the 
flask  is  about  half  full,  the  point  is  removed  from 
the  fluid,  and  the  contents  carefully  heated  over 
the  flame  and  allowed  to  boil  gently  for  a  couple  Fio  36  _Aut 
of  minutes.  While  the  steam  is  escaping  with  a  matic  suction- 
whistling  sound  from  the  end  of  the  tube,  the  ?ulbfor Collect- 

mg  Air-germs. 

flame  is  removed,  and  the  point  is  at  once  care- 
fully fused.  The  apparatus  is  now  ready  for  use,  for  which  it  is 
held  as  far  from  the  experimenter  as  possible,  in  the  direction 
from  which  the  wind  comes,  so  that  germs  shall  not  be  blown 
from  one's  body  toward  it.  The  point  is  opened  by  a  pair  of 
pincers  or  shears  that  have  been  flamed,  and  the  air  rushes  m 
with  a  whistle  and  fills  the  receptacle.  The  opening  is  once 
more  sealed,  the  flask  shaken  so  as  to  wash  all  germs  from 
the  walls  into  the  fluid,  and  the  development  of  micro-organ- 
isms in  the  fluid  is  awaited.  [But  this  gives  no  quantitative 
determination,  and  a  satisfactory  investigation  of  even  the 
aerobic  forms  present  in  the  fluid  is  only  possible  when  some 
of  the  fluid  after  thorough  shaking  is  at  once  taken,  with  the 
usual  precautions,  as  a  basis  for  some  form  of  plate-culture. 
—W.  T.I 


Bacteriological    Technology. 


Hesse's  aeroscope  (Fig-.  3?)  consists  of  a  glass  tube  50  to 
70  cm.  long-  and  4  cm.  in  diameter,  one  end  of  which  is  some- 
whab  flaring  and  closed  by  a  perforated  rubber  stopper  (d), 
A  sf|«  through  which  passes  a  short  glass  tube  10 

mm.  in  diameter,  plugged  with  cotton  (b).  The 
flange  at  the  other  end  is  somewhat  more  prom- 
inent; over  it  are  tied  two  thin  rubber  caps, 
one  outside  the  other,  the  inner  of  which  is 
pierced  with  a  round  hole  about  10  mm.  in 
diameter,  as  shown  in  the  section  in  Figure  37. 
The  tube  is  used  with  nutrient  gelatin,  the  sur- 
face of  which  must  not  reach  above  the  lower 
side  of  the  small  glass  tube  or  the  hole  in  the 
inner  cap,  when  the  apparatus  lies  flat. 

Tlie  tube,  stopper,  and  caps  are  cleansed  in 
1.0   per  cent   sublimate  solution,  and  finally 
rinsed  with  boiled  water.     The  inner  cap  is 
tied   fast   with  thread,   without  being-  much 
stretched.     If  it  is  found  to  be  water-tight,  by 
half  filling  the  tube  with  water,  and  holding- 
this  end  downward,  the  round  hole  (f  and  /') 
is  clipped  in  its  centre  with  sharp  scissors,  and 
the  outer  cap  is  tied  over  it,  but  in  a  more 
tense  condition.      [Rubber  caps,  fitting-  quite 
well  and  easily  applied,  are  also  supplied  with 
the  apparatus  by  dealers. — W.  T.]     Its  power 
to  hold  water  is  then  again  tested.     The  stop- 
per with  its  plugged  tube  in  place  is  now  in- 
serted in  the  other  end,  and  the  apparatus,  still 
containing  water,  is  hung  in  the  steam  cylin- 
der, which  needs  to  be  lengthened  for  this  pur- 
pose (Fig.  4.  C).     A  piece  of  wire  (Fig.  37,  c) 
twisted  around  the  upper  end  is  used  for  sus- 
pending  the    tube   to    the  lid  by  the  means 
shown  in   Figure   4   A.      After    exposure    to 
FIG.  37.— Hesse1*    streaming-  steam  for  a  quarter  of  an  hour,  it  is 
Aeroscope.        removed    from    the    sterilizing-  cylinder,   and 
when  it  is  somewhat    cooled  the  water  is  poured    out  and 
replaced  with  all  precautions  by  melted  sterile  nutrient  g-el- 
atin,  poured  from  a  pipette  orf  wash-bottle.     The  stopper  be- 
ing again  in  place,  it  is  once  more  hung  in  the  steam-cylinder 


72  Bacteriological    Technology. 

for  ten  minutes'  sterilization  at  100°,  after  which  it  is  placed 
horizontally  while  the  gelatin  cools  [or  rotated  until  the  gel- 
atin is  nearly  hard,  when  it  is  allowed  to  lie  flat  so  that  one 
part  of  the  circumference  is  more  thickly  coated  than  the  rest. 
— W.  T.]  When  wanted  for  use,  it  is  set  upon  a  suitable  sup- 
port, where  the  air  is  to  be  examined.  Hesse  uses  a  folding- 
tripod  such  as  is  used  by  photographers,  which  ends  in  a  flat 
surface  on  which  the  tube  can  be  fastened,  the  two  flasks  of 
the  aspirator  (Fig.  33)  being  hung  at  different  heights  on  the 
legs.  Simple  supports,  answering  every  purpose,  are  also 
easily  improvised.  When  the  apparatus  is  set  up,  it  is  con- 
nected with  the  aspirator,  the  surface  of  which  is  carefully 
washed  with  0.1  per  cent  sublimate,  and  the  aspirator  set  in 
operation,  the  siphon  being  sucked  full  beforehand,  to  prevent 
a  sudden  irregular  suction  when  the  pinch-cock  is  opened. 
The  quantity  of  air  which  Hesse  advises  to  be  passed  through 
the  apparatus  is  1  to  5  litres  for  inhabited  rooms,  and  10  to  20 
litres  out  of  doors,  but  in  certain  cases  much  greater  or  smaller 
quantities  may  be  advisable.  He  recommends  the  aspiration 
of  a  litre  of  air  through  the  apparatus  in  one  to  three  minutes 
for  inhabited  rooms,  and  the  same  quantity  in  three  to  four 
minutes  for  the  open  air,  as  a  proper  rapidity.  After  the  air 
has  been  sucked  through,  the  apparatus  is  closed  by  replacing 
the  rubber  membrane  (which  has  been  rinsed  in  sublimate), 
and  set  aside  at  room  temperature.  In  the  course  of  the  next 
eight  or  ten  days,  colonies  of  moulds  and  bacteria  grow  on  the 
surface  of  the  gelatin,  from  which  they  can  be  obtained  for 
examination  and  the  inoculation  of  cultures  by  using  a  long 
glass  rod  bearing  a  platinum  needle.  If  the  suction  has  been 
effected  sufficiently  uniformly  and  with  the  right  rapidity,  so 
that  all  germs  have  been  allowed  to  settle  from  the  air  passed 
through,  the  colonies  should  be  most  numerous  near  the  end 
through  which  the  air  enters,  gradually  decreasing  in  number 
toward  the  other  end,  the  last  third  or  quarter  of  the  gelatin 
surface  being  entirely  free  from  colonies. 

Bubbling  Apparatus. — Of  the  different  forms  with  fluid 
contents,  only  one,  long  employed  by  Miquel,  is  described  here. 
This  is  a  flask  closed  above  with  a  cap  plugged  with  cotton 
(a),  like  a  Pasteur  flask  (c/.  Fig.  10).  The  neck  is  prolonged 
as  a  fine  tube  to  the  bottom  of  the  flask.  The  slightly  con- 
stricted side  tube  b,  is  plugged  at  two  points  with  cotton  (not 


Bacteriological    Technology.  73 

shown  in  the  figure),  and  is  connected  with  some  form  of 
aspirator  by  the  rubber  tube  e.  A  capillary  tube  hermetically 
sealed  at  d,  -is  connected  by  the  same  means  with  the  other 
lateral  tube  (c),  which  is  slightly  bent  downward.  30  to  40 
cm.  of  distilled  water  is  poured  into  the  llask.  Sterilization 
is  effected  as  usual.  The  aspirator  is  connected  with  e,  the 
plugged  cap  a  is  flamed  and  removed,  the  observer  withdraws 
from  the  apparatus,  and  the  suction  of  air  into  it  is  allowed  to 
go  on,  after  which  the  cap  is  again  flamed  and  replaced.  By 
blowing  through  e  the  water  is  made  to  rise  and  fall  through 
the  neck  of  the  flask  ten  times,  to  wash  off  adhering  germs. 
The  point  d  is  flamed  and  broken  off  and  by  tipping  the  flask 
and  blowing  through  e  the  contents  are  divided  among  30  or 


d 

FIG.  38.-— Miquel's  Aeroscope. 

40  culture  flasks  containing  bouillon.  Finally,  the  outermost 
of  the  two  cotton  plugs  in  the  tube  b  is  removed  and  the  inner 
one  pushed  into  the  flask  with  a  sterilized  platinum  wire,  after 
25  cc.  of  sterile  bouillon  has  been  poured  into  the  flask.  The 
flask,  and  the  30  culture- vessels  are  set  aside  at  30°  C.,  and 
observed  for  at  least  a  month.  When  a  small  number  of  the 
vessels  show  growth,  the  total  of  the  germs  collected  by  the 
aeroscope  can  be  approximately  calculated  (c/.  p.  41,  method 
of  dilution). 

Tryde,  Hueppe,  and  v.  Sehlen  formerly  caused  air  to  bubble 
through  melted  nutrient  gelatin,  in  their  aeroscopic  analyses. 
Quite  recently,  Straus  and  Wurtz  have  used  the  same  method, 
and  for  this  purpose  produced  the  glass  apparatus  shown  in 
Figure  39.  The  top  and  bottom  measure  15  mm.  in  diameter, 
while  the  central  portion  (A)  is  a  wide  cylindrical  receptacle 
bearing  the  slightly  constricted  lateral  tube  (D),  with  two 


74 


Bacteriological    TecJmology. 


cotton  plug's  (f  and  g).  The  tube  B  is  drawn  out  to  a  fine 
point  below,  and  closed  above  with  a  cotton  plug-  (e).  At  c  it 
is  widened,  and  ground  to  fit  the  mouth  of  A  air-tight.  The 
apparatus  is  sterilized  at  150°  C.,  after  which  10  cc.  of  10  per 
cent  gelatin  (or  preferably  agar-gelatin)  are  poured  into  it 
and  a  drop  of  sterilized  olive-oil  added,  after  which  all  is  ster- 
ilized in  the  steam  cylinder.  The  addition  of  oil  prevents  the 
gelatin  from  foaming,  even  when  air  is  passed  through  it  very 
rapidly,  e.g.,  50  litres  in  ten  minutes.  The 
gelatin  is  melted,  D  is  joined  by  rubber-tubing 
with  an  aspirator,  and  the  plug  e  is  removed. 
While  air  is  passing  through,  the  gelatin  is 
kept  fluid  by  the  warmth  of  the  hand,  or,  if 
agar-gelatin  is  used,  a  water-bath  is  employed. 
Afterward,  the  plug  e  is  replaced,  and,  by  blow- 
ing into  D  the  fluid  is  forced  into  B  several 
times  to  wash  out  any  adhering  germs.  The 
plug  /  is  removed  so  as  to  allow  g  to  be  pushed 
into  the  gelatin  with  a  sterile  platinum  wire, 
after  which  it  is  replaced  and  g  is  gently 
shaken  about  in  the  gelatin,  which  is  then  al- 
lowed to  solidify  as  for  an  Esmarch  tube-cul- 
ture, or  poured  out  in  glass  trays  as  described 
on  pages  496,  497.  The  observation  of  the  de- 
velopment of  the  colonies  is  rendered  not  a  lit- 
tle difficult  by  the  fact  that,  while  air  is  bub- 
bling through,  the  oil  becomes  finely  emulsified 
in  the  gelatin,  rendering  it  cloudy. 

Insoluble  Powder  Filters. — As  the  result  FIG.  39.— Aeroscope  of 
of  a  series  of  very  carefully  conducted  experi-  strausandw»rtz- 
ments,  Petri  has  recommended  sand  as  a  filtration  medium 
in  preference  to  glass-wool  and  asbestos,  which  have  been 
used  by  others  (Miquel,  Moreau,  Freudenreich,  and  Frank- 
land).  The  details  of  his  method  are  as  follows:  sand  is 
passed  through  a  sieve  with  meshes  0.5  mm.  wide,  and  what 
passes  this  is  sifted  through  a  second  with  meshes  0.25  mm. 
wide,  the  portion  that  remains  in  this  consisting  of  grains 
of  the  right  size.  This  sand  is  heated  to  redness  in  an  iron 
crucible  (half  or  three-quarters  of  an  hour  being  required 
for  100  cc.),  while  it  is  stirred  with  a  glass  rod.  While 
warm,  it  is  filled  into  sterile  test-tubes  plugged  with  cot- 


Bacteriological    Technology. 


75 


are  brought 


ton.  Two  sand-filters  (s\  and  s2)  3  cm.  long 
into  a  glass  tube  1.5  to  1.8  cm.  wide  and  9  cm.  long,  as  shown 
in  Figure  40.  The  sand  is  held  in  place  by  small  caps  (n\  to 
7i4)  of  fine  brass  gauze  (40  meshes  to  the  linear  centimetre), 
which  are  easily  made  over  the  end  of  a  glass  tube  or  metallic 
rod.  This  separation  of  the  sand  in  two  parts  enables  the  ex- 
perimenter to  sow  the  second  sand  plug  separately,  as  a  con- 
trol filter.  The  filters  being  1  cm.  from  each  end  of  the  tube, 
it  is  plugged  at/  and  g  with  cotton,  and  sterilized  at  150°  C. 
Before  use,  the  cotton  plugs  are  removed  and  laid 
aside  in  a  sterilized  glass  box.  The  filter-tube  is 
joined  with  an  aspirator  by  means  of  a  rubber 
stopper  (a)  sterilized  in  sublimate,  pierced  for  a 
cotton-plugged  sterile  glass  tube  c,  which  is  con- 
nected by  a  short  piece  of  rubber  tubing  with  a 
lead  tube  (e)  0.5  cm.  wide.  The  filter  tube  being 
arranged  vertically,  the  cotton  plug  is  removed 
from  g  and  the  suction  can  begin.  Petri  uses 
as  an  aspirator  a  hand  air-pump,  each  stroke  of 
which  draws  a  litre  of  air  through  the  filter. 
When,  as  in  his  experiments,  100  litres  pass 
through  the  filter  in  10  to  20  minutes,  all  germs 
are  stopped  by  the  first  filter.  The  material  is 
sown  in  pairs  of  trays  (Fig.  12)  9  cm.  in  diameter, 
the  sand  of  the  first  filter  being  divided  between 
three,  and  of  the  second,  between  two  such  tra3rs. 
The  sand  is  first  poured  into  the  trays,  and 
melted  gelatin  is  poured  over  it  from  test-tubes 
(cf.  p.  57).  After  the  sand  is  completely  wet  by 
the  gelatin,  so  that  all  air  bubbles  have  been 
driven  out,  it  is  equally  distributed  by  a  series  of 
short  but  forcible  horizontal  movements,  and  the  gelatin  is 
allowed  to  harden,  when  the  three  pairs  of  trays  are  set  away 
one  over  the  other  at  the  temperature  of  the  room. 

Soluble  Filters. — Miquel,  the  most  experienced  living  stu- 
dent of  the  air,  has  very  recently  recommended 5  the  employ- 
ment of  soluble  filters,  an  ingenious  method  suggested  by  Pas- 
teur over  twenty-five  years  ago.  Of  the  various  powders 
which  may  be  selected  (cane-sugar,  table-salt,  sodium  phos- 
phate, magnesium  sulphate,  etc.),  Miquel  recommends  cane- 
sugar,  which  demands  no  special  preparation,  may  be  steril- 


FIG.  40.— Petri's 
Sand- Filter. 


76 


Bacteriological    Technology. 


ized  at  150°  C.,  without  losing-  its  high  degree  of  solubility  or 
becoming  more  hygroscopic,  or  without  having  any  injurious 
effect  on  the  bacteria.  In  very  foggy  weather  it  may  become 
impossible  to  work  with  either  insoluble  or  soluble  filters.  In 
this  case  a  bubbling  apparatus  may  occasionally  have  to  be 
used.  The  sugar  (if  loaf  sugar  is  used)  is 
powdered  in  a  mortar  and  shaken  through 
two  metal  sieves,  as  described  for  sand,  so 
that  the  size  of  the  grains  used  is  about  half 
a  millimetre.  This  sugar  is  placed  in  an 
aeroscope  (Fig.  41,  A)  consisting-  of  a  glass 
tube  about  20  cm.  long  and  5  mm.  in  diam- 
eter, narrowed  at  e,  and,  according-  to  the 
suggestion  of  Freudenreich,  provided  at  b 
with  a  ground  cap  plugged  with  cotton,  as 
for  the  Pasteur- Chamberland  flasks.  Two 
plugs  of  (glass-wool  or)  cotton  are  inserted 
at  d  and  /,  and  the  whole  is  sterilized  at  150° 
C.  The  cap  is  removed,  and  enough  well- 
dried  sugar  (1  to  2  gm.)  is  poured  into  the 
tube  to  fill  it  for  a  length  of  8  to  10  cm.,  after 
which  all  is  again  sterilized  at  150°  C.  When 
it  is  to  be  used,  by  a  series  of  light  percussions 
the  sugar  is  packed  against  the  plug  d,  and 
the  tube  held  nearly  vertically  (tipped  toward 
the  wind),  so  that  during  suction  the  sugar 
may  not  fall  away  from  the  walls,  thus  render- 
ing it  possible  for  the  air  to  pass  unfiltered  at 
any  point. 

The  air  is  drawn  through  the  filter  Vith 
different  rapidity  and  for  a  varying  length  of 


..e 


A. 


B, 


time,  according  to  circumstances.     The  aver-    FIG.  41.— A,  Newer,  B, 
age  number  of  sperms  for  the  day  at  a  given  Older  Model  of  Miquers 

J  Soluble  Powder  Filter. 

point  is  obtained  by  slowly  drawing  the  air 
through  a  filter  for  12  or  24  hours.     To  learn  the  number 
at  a  given  time,  it  is  necessary  to  draw  the  largest  possi- 
ble quantity  of  air  through  the  sugar  in  the  shortest  time 
possible. 

After  the  aspiration,  the  cotton  or  glass-wool  plug  is  re- 
moved and  the  sugar  is  poured  into  sterilized  water.  When 
many  germs  are  expected,  a  large  quantity  of  water  (500  to 


Bacteriological    Technology.  77 

1,000  cc.)  is  used;  otherwise  a  smaller  quality  (50  to  100  cc.) 
is  taken. 

In  case  the  sugar  does  not  run  out  of  the  tube  easily,  it 
must  be  pushed  out  by  the  aid  of  the  cotton  plug  /,  and  a 
sterile  glass  rod  introduced  at  /,  and  the  tube  afterward  rinsed 
out  with  sterile  water.  The  plug  d  is  also  to  be  sown  sepa- 
rately as  a  test  of  the  efficiency  of  the  filter,  which  should 
remain  free  from  germs  after  the  air  has  been  sucked  through. 
To  simplify  these  manipulations,  I  advise  the  use  of  tubes 
that  are  not  constricted  at  e,  and  to  proceed  as  follows :  The 
following  articles  are  to  be  made  ready  beforehand:  1,  an 
Erlenmeyer  flask,  filled  with  sterile  water;  2,  a  short  test-tube, 
with  a  few  cc.  of  water ;  3,  a  test-tube  with  nutrient  gelatin ; 
4,  a  sterile  glass  box  (Fig.  11  or  13);  5,  a  small  rubber  tube. 
The  plug  /  is  removed  with  forceps,  and  laid  in  the  glass  box, 
after  which  d  is  similarly  removed  and  cast  quickly  into  the 
gelatin,  /  being  immediately  replaced.  The  cap  a  is  then  taken 
off,  and  the  sugar  poured  out  into  the  Erlenmeyer  flask,  the 
rubber  tube  is  slipped  over  the  unground  end  of  the  glass  (/). 
the  ground  end  being  dipped  into  the  sterile  water  of  the  short 
test-tube,  when  all  remaining  germs  and  sugar  are  rinsed 
out  by  alternately  sucking  water  into  the  tube  and  expel- 
ling it;  finally  this  water  is  poured  into  an  Erlenmeyer  flask. 
The  filter  tubes  which  Miquel  used  in  his  earlier  investigations 
were  drawn  out  to  a  capillary  point  at  the  end  through  wrhich 
air  was  admitted  (Fig.  41  B,  \vhere  the  constriction  between 
the  two  cotton  plugs  is  also  left  out),  and  were  opened  and 
closed  by  breaking  off  and  remelting  the  point.  The  use  of 
the  ground  cap  a  renders  it  far  easier  to  empty  the  apparatus, 
but  adds  materially  to  its  expense  ;  there  is,  however,  no  rea- 
son why  simple  glass  tubes,  closed  by  my  rubber-tubing  cap 
(cf.  Fig.  9,  III.-VI.)  should  not  be  used.  The  gelatin  in  which 
the  plug  d  was  placed  should  remain  sterile,  all  germs  being- 
found  in  the  Erlenmeyer  flasks.  When  the  sugar  is  entirely 
dissolved  and  the  solution  well  shaken,  the  number  of  germs 
is  determined  exactly  as  described  for  water  analysis,  prefer- 
ably using  agar-gelatin,  so  as  to  avoid  as  far  as  possible  the 
disturbing  liquefaction  of  the  gelatin. 

A  great  advantage  of  this  method  is  that  only  a  fraction 
of  the  sugar  solution  and  the  germs  it  contains  need  be  used 
for  quantitative  analysis,  while  the  greater  part  can  be  used 


yS  Bacteriological    Technology. 

for  various  qualitative  investigations  (e.g.,  a  search  for  anae- 
robic forms),  which  it  was  not  possible  to  carry  out  in  the 
earlier  methods  in  which  all  of  the  collected  germs  had  to  be 
used  for  the  quantitative  analyses. 

Of  the  aeroscopes  and  methods  of  analysis  here  described, 
the  first  two  have  really  only  historical  interest.  The  auto- 
matic suction  balloon  was  the  instrument  with  which  Pasteur 
long  since  outlined  the  bacteriological  topography  of  the  at- 
mosphere, which  Miquel  has  since  persistently  worked  upon. 
Hesse's  apparatus  marks  the  first  attempt  to  employ  the 
gelatin  cultures  of  Koch  in  quantitative  analysis  of  the  air. 
Of  the  other  four,  doubtless  the  solid  filters  will  deserve  pref- 
erence. They  are  easily  transported,  and  can  be  "  charged  " 
with  bacteria  at  any  temperature  and  at  places  far  from  the 
laboratory,  after  which  they  can  be  kept  (at  least  for  several 
days)  until  cultures  can  be  started  under  favorable  conditions. 
They  also  accomplish  what  is  possible  toward  collecting  in  a 
relatively  short  time  all  germs  from  the  air  of  a  very  large 
space.  Of  these  solid  filters,  the  soluble  appear  to  me  far 
superior  to  the  insoluble. 

In  this  chapter,  we  have  as  good  as  exclusively  concerned 
ourselves  with  the  application  of  Koch's  methods  of  isolation 
in  gelatin  to  bacteriological  analysis.  It  must,  also,  be  said 
unqualifiedly  to  be  the  chief  method  of  such  analysis.  Start- 
ing plate-cultures  in  one  or  other  of  the  described  'forms  is  the 
means  first  to  be  employed  when  bacteria  are  to  be  found  and 
separately  cultivated  from  a  given  substance,  and  they  will 
in  most  cases  lead  to  the  desired  result  if  sufficient  attention 
is  given  to  varying  the  culture-material,  temperature,  time  of 
observation,  number  of  germs,  etc.  The  later  the  colonies  are 
counted,  the  more  probability  will  there  be  that  all  germs 
have  developed  into  visible  colonies.  It  is  not  so  much  the 
danger  of  atmospheric  contamination  which  leads  to  an  earlier 
examination  than  might  be  wished,  as  the  presence  of  rapidly 
growing,  liquefying  bacteria  and  moulds,  which  quickly  spread, 
so  as  to  cover  large  portions  of  the  gelatin.  But  it  is  self- 
evident  that  it  is  not  a  universal  method  in  the  sense  of  ren- 
lering  every  other  method  superfluous.  On  the  contrary,  we 
may  often  do  .well  to  use  the  various  other  methods  described 
in  Chapter  IV.,  either  alone  or  as  complementary;  but  no 


Bacteriological    Technology.  79 

general  rules  can  be  given  as  to  this,  so  that  one  must  choose 
his  plan  for  each  respective  case. 

Finally,  a  most  important  point  must  be  noted  with  refer- 
ence to  all  the  methods  thus  far  described,  viz. :  They  are 
calculated  for  only  aerobic  bacteria.  An  exhaustive  analysis 
of  an  unknown  mixture  of  bacteria,  includes  also  a  determin- 
ation of  the  pronouncedly  anaerobic  forms,  which  play  so  im- 
portant a  part  in  the  economy  of  nature,  as  well  as  in  the 
causation  of  disease.  In  what  precedes,  these  have  been  left 
entirely  out  of  consideration,  being-  reserved  for  special  and 
•detailed  discussion  in  the  next  chapter. 


CHAPTEE  YIII. 

CULTURE    OF  ANAEROBIC  BACTERIA. 

IN  1861,  Pasteur  made  the  surprising-  discovery  that  organ- 
isms exist  which  can  live,  nourish  themselves,  and  propagate, 
without  access  to  free  oxygen.  His  doctrine  of  anaerobiosis 
("la  vie  sans  air")  was  received  with  doubt  and  mistrust;  but 
further  investigation  not  only  corroborated  his  observation, 
but  from  this  as  a  starting  point,  Pasteur  succeeded  in  lead- 
ing the  physiology  of  respiration  into  new  channels,  bringing 
us  nearer  to  an  understanding  of  many  important  processes 
of  fermentation.  Anaerobiosis  showed  itself  to  be  a  very  sig- 
nificant and  wide-spread  phenomenon,  and  a  closer  investiga- 
tion of  the  need  for  oxygen  of  different  bacteria  (as  well  as 
yeasts  and  moulds)  revealed  an  extreme  variability  in  this 
respect.  Some  bacteria  demand  a  very  abundant  supply  of 
oxygen,  while  others  are  affected  by  it  as  a  poison,  which  not 
only  hinders  their  development,  but  quickly  destroys  their 
life.  Between  these  extremes— the  marked  aerobiotic  and 
anaerobiotic  forms — every  degree  of  transition  is  to  be  found. 
Some  forms  seem  to  thrive  about  equally  well  with  or  without 
free  oxygen;  some  grow  freely  exposed  to  oxygen  of  a  low 
tension,  others  when  this  is  greater — a  circumstance  which 
occasionally  finds  expression  in  a  very  striking  manner  in  the 
form  and  appearance  of  gelatin  cultures.  While  the  growth 
around  the  puncture  matte  in  the  gelatin  occurs  for  many 
species  very  abundantly  at  and  immediately  below  the  surface, 
and  diminishes  toward  the  bottom  (Fig.  42,  c),  other  cultures 
may  be  found  (Fig.  42,  a)  which  show  an  extremely  slight 
growth  near  the  surface,  while  the  colonies  of  bacteria  increase 
uniformly  in  size  the  further  they  are  removed  from  the  at- 
mosphere. The  former  is  consequently  aerobic,  the  latter 
anaerobic.  Beside  these  more  or  less  conical  cultures,  with 
the  point  directed  downward  or  upward,  others  occur  as  capil- 


Bacteriological    Technology. 


81 


lary  or  thick  cylinders  along-  the  needle  puncture  (Fig.  42,  6). 
The  oxygen  of  the  air  has  neither  helped  nor  hindered  the 
growth  of  these. 

The  discovery  of  anaerobiosis  has  necessitated  the  intro- 
duction of  new  apparatus  and  methods  in  the  cultivation  of 
bacteria.  These  must  be  treated  now  in  detail,  especially 
since  we  already  know  several  pathogenic  forms  which  are 
markedly  anaerobic  (malignant  oedema,  charbon  symptoma^ 
tique).  Very  different  means  have  been  employed  for  keep- 
ing the  oxygen  of  the  air  from  culture  vessels  and  material, 


FIG.  42.— Test-tube  Cultures,  a,  B.  murisepticus,  in  agar-gelatin ;  6,  a  colorless  yeast  in 
raisin-gelatin;  c,  B.  Anthracis,  in  agar-gelatin.  The  three  cultures  are  of  like  age,  and 
grown  under  exactly  the  same  condition? 

or  for  removing  all  absorbed  oxygen  from  the  latter.  Some- 
times the  effort  has  been  limited  to  covering  the  culture- 
medium  with  solid  substances  (glass,  mica,  ag-ar-agar),  fluid 
(oil),  or  g-as  (carbonic  acid,  hydrogen).  If  g-ood  results  are  de- 
sired, the  free  oxygen  held  by  the  culture-medium  must  also 
be  removed  by  boiling-,  the  use  of  the  air-pump,  the  passage 
of  a  g-as  which  is  not  a  supporter  of  combustion,  but  at  the 
same  time  not  poisonous  (hydrogen),  or  by  the  aid  of  aerobic 
bacteria  or  chemicals  with  an  affinity  for  oxyg-en. 

Examples  of  most  of  these  methods  are  described  below, 
6 


82  Bacteriological    Technology. 

* 

though  I  shall  confine  myself  to  a  discussion  of  a  limited  num- 
ber of  kinds  of  apparatus  which  have  received  the  recommen- 
dation of  expert  investigators.  In  accordance  with  the  plan 
of  the  book,  the  principal  weight  will  then  be  laid  on  such 
methods  as  demand  the  most  readily  obtainable  and  cheapest 
material.  Those  who  wish  to  go  further  into  the  technology 
of  this  branch  of  the  subject  are  referred  to  the  two  chief 
treatises  on  the  subject,  by  Roux 6  and  Siborius.7 

Even  by  the  best  of  the  methods  described,  it  is  not  possi- 
ble to  remove  the  last  trace  of  oxygen  from  the  culture- 
vessels,  but  they  have  shown  themselves  sufficiently  free  from 
oxygen  under  all  circumstances  for  the  cultivation  of  the  most 
pronouncedly  anaerobic  forms.  As  an  easily  applied  test  for 
freedom  from  oxygen,  sufficiently  accurate  for  our  purpose, 
indigotin  is  to  be  recommended.  Enough  of  a  very  dilute 
solution  of  the  sulphate  (1  gm.  indigotin  to  a  litre  of  water)  is 
added  to  the  culture -medium  to  give  it  a  distinct  blue  color,  a 
small  quantity  of  grape  sugar  is  added,  and  the  reaction  ren- 
dered distinctly  alkaline  by  the  addition  of  potash.  Three 
drops  of  a  10-per-cent  solution  of  potash  are  added  to  10  cc.  of 
nutrient  gelatin  or  agar,  which  in  addition  to  the  usual  sub- 
stances also  contains  1  per  cent  of  grape  sugar  (Liborius). 
When  (by  means  of  boiling,  the  passage  of  hydrogen,  or  the 
use  of  the  air-pump)  the  oxygen  absorbed  by  the  blue  culture 
substance  is  removed,  the  latter  becomes  colorless,  since  the 
indigo-blue  is  reduced  by  the  grape  sugar  to  indigo-white.  A 
return  of  the  blue  color  shows  that  oxygen  is  not  certainly 
enough  excluded. 

Trustworthy  and  adequate  apparatus  for  the  cultivation 
of  anaerobic  forms  can  only  be  prepared  when  a  hydrogen 
developer  or  air-pump  can  be  used.  For  the  former,  Kipp's 
apparatus  is  commonly  used,  though,  as  Jorgensen  has  shown, 
any  one  can  fit  up  a  sufficiently  good  generator  at  a  cost  of 
about  half  a  dollar,  by  using  a  common  student-lamp  chimney, 
fitted  by  a  loose  cork  (Fig.  43,  a)  into  a  cylindrical  glass  which 
contains  a  mixture  of  one  part  by  weight  of  sulphuric  acid 
and  eight  parts  by  weight  of  water  (prepared  with  care  by 
adding  the  acid  to  the  water  while  it  is  being  stirred, — never 
by  adding  water  to  the  acid),  and  a  couple  of  drops  of  plati- 
num chloride  solution.  A  perforated  lead  plate  covered  with 
muslin  is  placed  at  fr,  to  support  a  quantity  of  granulated 


Bacteriological    Technology. 


zinc  in  pieces  about  as  large  as  peas.  The  stopper  c  must 
be  of  rubber,  and  fit  perfectly.  Hydrogen  is  evolved  as 
soon  as  the  zinc  is  brought  in  contact  with  the  acid;  but  if 
the  rubber  tube  at  d  is  closed  by  a  pinch-cock,  the  hydrogen 
drives  the  acid  down  to  below  6,  and  its  formation  stops,  to 
recommence  when  the  pinch-cock  is  again  loosened.  On  the 
way  to  the  culture-vessels,  the  gas  is  passed  through  an  alka- 
line solution  of  pyrogallic  acid  (e)  (1  part  of  a  25-per-cent 
aqueous  solution  of  pyrogallic  acid  is  thoroughly  mingled,  in 

a  perfectly  full  bottle,  with  a 
60-per-cent  solution  of  potash) 
to  remove  a  trace  of  oxygen 
which  may  be  present. 

The  air-pump  belongs  to  the 
class  of  expensive  instruments 
which  it  is  not  proposed  to  dis- 
cuss here  in  detail.  It  is,  in- 
deed, a  great  comfort  to  have 
the  use  of  a  mercurial  air-pump 
when  the  cultivation  of  anaero- 
bic forms  is  to  be  undertaken, 
but  it  is  not  a  necessity.  A 
comparatively  cheap  and  good 
model  is  the  Sprengel  pump,  as 
modified  by  Hiifner.  One  of 
the  simple  water  air-pumps 
cannot  be  relied  on  for  the  re- 

Qf  oxyg.en  ag  completely 


FiG.43.-.Toergensen's  Hydrogen-generator. 

as  is  sometimes  necessary;  but  by  also  employing  hydrogen 
as  indicated  by  Roux  (infra,  p.  87),  it  may  be  effected  with 
sufficient  completeness. 

The  vessels  and  methods  for  cultivating  anaerobic  forms 
will  be  considered  under  two  principal  classes,  according  as 
they  are  to  be  used  for  isolating  and  obtaining  pure  cultures 
of  anaerobic  forms  from  a  mixture  of  bacteria,  or  merely  for 
propagating  a  species  already  pure,  either  in  culture  or  in  a 
diseased  animal. 

I.  ISOLATION  FROM  A  MIXTURE  OF  SPECIES. 

Pasteur  succeeded  in  getting  the  first  pure  cultures  of  an- 
aerobic forms  in  fluid  media,  by  the  method  of  isolation  based 


84  Bacteriological    Technology. 

on  physiological  differences  of  species,  using-  carbonic  acid  or 
exhausting-  the  air.  More  recently,  the  attempt  has  been 
especially  made  to  apply  Koch's  methods  with  gelatinized 
media,  even  to  the  analysis  of  mixtures  containing-  anaerobic 
bacteria. 

A.  Simpler  (and  more  incomplete)  means,  which  do  not 
.require  the  use  of  either  air-pump  or  hydrogen  generator. 

1.  Plate  Cultures  under  Mica  (Koch).— Koch  made  the  at- 
tempt to  effect  the  plate-culture  of  anaerobic  forms  by  cover- 
ing the  gelatin,  while  still  soft,  with  a  sterile  sheet  of  mica. 
This  must  usually  be  as  thin  as  a  sheet  of  paper,  without 
flaws,  and  sterilized  by  flaming  immediately  before  use  (or  at 
150°  C.,  wrapped  in  paper),  and  care  is  to  be  taken  not  to  allow 
air-bubbles  to  slip  under  it  when  it  is  laid  on  the  gelatin.     To 
secure  a  still  surer  exclusion  of  oxygen,  the  margin  of  the 
mica  can  be  covered  with  melted  paraffin  (Fraenkel),  which 
quickly  hardens,  forming  a  solid  border.     In  this  way,  the 
usual  method  of  working  is  preserved,  as  well  as  an  easy  ac- 
cessibility for  microscopic  examination. 

It  is  easy  to  convince  one's  self  that  the  mica  plate  really 
hinders  the  access  of  oxygen  to  germs  beneath  it,  to  no  small 
degree.  If  a  decidedly  aerobic  non-liquefying  form  has  been 
plated  out  in  this  manner,  the  colonies  may  be  seen  to  appear 
in  numbers  in  the  uncovered  part  of  the  gelatin,  while  beneath 
the  mica  they  occur  sparingly  near  the  margin,  and  in  the 
beginning  may  be  entirely  wanting  in  the  centre.  Yet  the 
method  is  frequently  unsatisfactory,  because  the  exclusion  of 
oxygen  is  not  sufficiently  complete  and  lasting,  and  it  is  infe- 
rior to  those  described  below,  especially  that  recommended  by 
Liborius. 

2.  Isolation  in  a  Very  Deep  Solid  Medium  (Liborius).— 
A  small  quantity  of  the  mixture  to  be  analyzed  is  sown  in  a. 
test-tube  filled  to  a  depth  of  10  to  20  cm.  with  nutrient  gelatin 
or  agar,  which  has  been  melted  and  cooled  to  the  lowest  tem- 
perature at  which  it  will  remain  fluid.     The  material  is  distri- 
buted as  uniformly  as  possible  by  aid  of  a  thin  sterile  glass  rod 
carried  carefully  through  a  rotary  as  well  as  vertical  move- 
ment in  the  gelatin.     If  the  distribution  is  successful,  and  the 
number  of  germs  right,  very  pretty  results  may  be  obtained, 
the  number  of  aerobic  forms  decreasing  in  size  and  number 
from  the  top,  while  on  the  other  hand  the  anaerobic  forms 


Bacteriological    Technology. 


fe 


grow  only  toward  the  bottom,  and  are  always  wanting-  from 
the  uppermost  part  of  the  gelatin.  In  either  case,  the  distri- 
bution of  the  colonies  always  gives  a  good  basis  for  deciding 
as  to  their  need  of  oxygen. 

The  colonies  of  bacteria  are  naturally  far  less  easily  acces- 
sible for  examination  in  the  depth  of  such  a  test-tube  than  in 
a  plate-culture.  When  there  are  few  of  them, 
and  each  colony  is  large  and  liquefies  the  gela- 
tin, material  for  new  cultures  or  for  examina- 
tion is  easily  obtained  by  thrusting  a  sterile 
capillary  tube,  open  below  but  sealed  at  top, 
through  the  gelatin  until  the  colony  is  reached, 
when  the  top  is  broken  off.  In  other  cases, 
it  may  be  necessary  to  break  the  tube  and  to 
cut  the  gelatin  up  in  a  sterile  tray  so  as  to 
render  the  colonies  accessible  to  the  needle. 
If  agar  is  used,  this  can  usually  be  blown  out 
of  the  tube  by  means  of  a  Pasteur  pipette  (p. 
44)  passed  down  at  one  side.  The  micro- 
scopic examination  of  the  colonies  under  mod- 
erately high  powers  can  likewise  often  be  con- 
veniently made  only  after  removing  the  con- 
tents from  the  glass  and  cutting  it  in  thin 
slices. 

B.  Methods  in  which  hydrogen  is  used  to 
replace  the  air. 
3.  Isolation  in  Gelatinized  Media. — Roux  recommends  a 
tube  similar  in  all  essentials  to  that  shown  below  in  Figure  5G, 
but  of  considerably  larger  size.  After  hydrogen  has  been 
passed  for  a  sufficiently  long  time  through  the  liquefied  and 
inoculated  gelatin,  this  is  allowed  to  solidify  while  the  tube  is 
horizontal.  When  the  colonies  are  developed,  the  tube  is  cut 
lengthwise  with  a  diamond  into  two  boat-shaped  halves,  the 
one  containing  the  gelatin  being  then  easily  accessible  for 
observation. 

Fraenkel  uses  the  following  method :  The  test-tubes,  rather 
thick -walled,  of  large  diameter,  and  without  spreading  rims, 
are  plugged  with  cotton  sterilized  in  the  usual  way,  and  inoc- 
ulated in  three  degrees  of  dilution  as  described  on  p.  500,  after 
which  the  plugs  are  replaced  as  rapidly  as  possible,  with  ster- 
ilized fingers,  by  closely  fitting  rubber  stoppers  with  double 


FIG.  44.— Isolation- 
Culture  of  Anaerobic 
Bacteria  in  a  Deep 
layer  of  Gelatin.— 
(Liborius.) 


86  Bacteriological    Technology. 

perforation,  containing-  two  glass  tubes  bent  at  right  angles 
above  the  stopper.  One  of  these  reaches  the  bottom  of  the 
test-tube,  while  the  other  ends  immediately  below  the  stopper. 
The  horizontal  branches  are  contracted  to  capillary  cavities 
near  the  ends,  which  are  plugged  with  cotton.  The  stopper 
and  tubing  must  be  carefully  sterilized,  before  the  test-tube  is 
infected.  This  is  done  exactly  as  described  above  (Chamber- 
land  filter,  p.  10),  i.e.,  by  sterilizing  the  stopper  in  0.1  per  cent 
sublimate,  and  the  tubes  at  150°  C.,  both  being  wrapped  in 
paper  and  finally  sterilized  by  steam  after  being  set  up  in  an 
autoclave;  the  stopper  and  tubing  can,  of  course,  be  quickly 
sterilized  together.  Just  before  being  used,  the  rubber  stopper 
is  unwrapped,  the  long  tube  drawn  through  the  flame,  to  be 
sure  of  its  sterilization,  and  the  stopper  firmly  fixed  in  the 
test-tube,  after  which  it  is  coated  with  paraffin,  especially 
about  the  small  tubes  and  where  it  meets  the  side  of  the  test- 
tube.  The  long  tube  is  now  connected  with  the  hydrogen 
generator,  while  the  test-tube  is  kept  at  37°  C.  by  the  use  of  a 
water-bath.  According  to  Fraenkel,  the  rapid  passage  of 
hydrogen  for  four  minutes  insures  the  removal  of  free  oxygen, 
after  which  the  two  tubes  are  sealed  by  melting  at  the  con- 
strictions, and  the  test-tube  is  rotated  under  a  stream  of  cold 
water  until  the  gelatin  has  solidified  upon  the  walls.  By  this 
means  a  "roll-culture"  of  anaerobic  forms  is  ultimately  ob- 
tained (Fig.  45). 

4.  The  Capillary  Tube  Method. — This  is  obviously  applic- 
able to  the  isolation  of  anaerobic  species,  and  has  been  espe- 
cially recommended  for  this  purpose  by  Klebs  and  Yignal.  The 
latter  advises  its  combination  with  the  passage  of  hydrogen 
and  the  use  of  solid  culture-media.  A  small  quantity  of  nutri- 
ent agar  or  gelatin  is  brought  to  the  boiling  poin  t  and  allowed 
to  cool  off  while  a  constant  stream  of  hydrogen  is  passed 
through  it  by  means  of  a  bent  Pasteur  pipette  inserted  be- 
tween the  wall  of  the  test-tube  and  its  cotton  plug  (Fig.  46). 
By  use  of  a  water  bath  the  gelatinized  contents  are  prevented 
from  solidifying.  The  tube  is  infected  while  hydrogen  is  pass- 
ing through.  After  this  has  been  continued  a  few  minutes 
longer,  and  while  the  stream  is  still  passing,  the  gelatin  is 
quickly  sucked  up  into  capillary  tubes  about  50  cm.  long, 
which  are  sealed  at  both  ends  and  can  be  fastened  later  upon 
black  cardboard. 


Bacteriological    Technology. 


C.  Methods  depending-  upon  the  use  of  the  air-pump. 

Where  an  air-pump  can  be  had,  a  very  simple  culture- 
apparatus  (Roux,  Fig-.  47)  can  be  used.  This  is  closed  by  a 
cotton  plug  and  contains  a  rather  small  quantity  of  nutrient 
gelatin.  Afer  the  latter  is  infected,  the  plug-  (a)  is  crowded 
down  to  the  constriction  c,  the  tube  is  softened  and  slightly 
constricted  at  b,  and  the  g-elatin  remelted.  By  means  of  a 
piece  of  rubber  tubing-,  the  tube  is  connected  with  the  air- 
pump,  and  the  air  exhausted  from  it,  the  glass  being  mean- 


FIG.  45. 


FIG.  46. 


FIG.  45.— Roll-culture  of  Anaerobic  Bacteria  (Fraenkel). 

FIG.  46.— Use  of  a  Bent  Pasteur  Pipette  for  Passing  Hydrogen  through  Gelatin  Prepara- 
tory to  Making  Capillary  Tube-cultures 

time  carefully  warmed  by  passing  a  g-as  or  spirit  flame  up  and 
down  its  sides  at  short  intervals.  When  the  air  is  exhausted, 
the  tube  is  melted  at  6,  which  hermetically  seals  it  and  at  the 
same  time  separates  it  from  the  air-pump.  A  "  roll-culture  " 
is  finally  obtained  by  allowing-  the  gelatin  to  harden  in  a  thin 
layer  on  the  inside  of  the  tube. 

According  to  Roux,  a  small  water  air-pump  (filter-pump) 
is  sufficient  if,  by  means  of  a  T-tube  with  two  glass  stop-cocks 
(Fig.  48),  the  culture-tube  is  likewise  joined  to  a  hydrogen 


88 


Bacteriological    TccJinology. 


generator,  and  the  exhaustion  of  the  air  alternated  five  or  six 
times  with  the  admission  of  hydrogen. 

Having-  now  explained  how  pure  material  of  anaerobic 

V.LJ* 


a 


FIG.  48. 


FIG.  49. 


Fio.47.— Culture-vessel  for  Isolating  Anaerobic  Bacteria  in  Gelatin  in  a  Vacuum  (Roux). 
FIG.  48. — The  Same   Apparatus  Arranged  with  T-tube  for  Alternating  Connection  with 
Filter-pump  and  Hydrogen-generator  (Roux). 

FIG.  49.— Vessel  for  Cultivation  of  Anaerobic  Species  under  Hydrogen  (Salomonsen). 

forms  is  obtained  by  cultures  in  solid  media,  it  remains  briefvy 
to  explain  the  use  of  fluid  media  for  the  same  purpose.     Three 


Bacteriological    Technology. 


courses  are  open,  so  that  it  is  possible  to  employ  the  capillary- 
tube  method,  as  described  on  p.  86,  or  the  method  of  dilution, 
by  adding-  a  drop  of  the  greatly  diluted  substance  to  each  of  a 
large  number  of  culture-flasks,  which  may  then  be  used  for 
cultures  under  hydrogen  or  in  a  vacuum. 

For  the  passage  of  hydrogen,  culture-vessels  like  that  of 
Fraenkel  (Fig.  43)  may  be  used,  or  my  own  (Fig.  49),  in  which 
the  part  used  for  the  culture  is  about  10  cm.  long"  and  2  cm. 
in  diameter.  The  contents  are  inoculated  through  the  verti- 
cal tube,  closed  at  top  by  a  cotton-plugg-ed  rubber  tube  (a), 


J, 


a 


FIG.  50.  FIG.  51. 

FIGS.  50  and  51.— Two  Culture- vessels  after  Pasteur. 

and  hydrogen  is  admitted  through  the  bent  tube,  provided  at 
d  with  an  ordinary  cotton  plug.  Just  behind  each  plug",  the 
tubes  are  narrowly  constricted  just  before  the  hydrogen  is 
passed  through,  and  as  soon  as  the  stream  is  discontinued  the 
apparatus  is  hermetically  sealed  at  these  points.  If  the  tubes 
are  made  long-  enough  in  the  first  place,  these  vessels  may  be 
used  several  times  in  succession. 

Vacuum-cultures  may  be  made  in  Roux's  tubes  (Fig.  47),  or 
in  those  shown  in  Figures  50  and  51,  which  have  been  con- 


Bacteriological    Technology. 


stantly  used  in  Pasteur's  laboratory  for  many  years.  These 
are  filled  through  the  capillary  tubes  (a),  by  applying  suction 
at  6,  after  which  the  fine  tubes  are  hermetically  sealed  and 
the  hole  sterilized.  The  contents  are  inoculated  by  sucking 
some  of  the  questionable  material  in  through  the  tube  a,  after 
this  has  been  heated  to  redness  and  bent  away  from  the  larger 
tube,  and  its  end  flamed  and  broken  off,  to  be  subsequently 
melted  together  again.  The  apparatus  is  connected  with  the 
air-pump  by  means  of  rubber  tubing.  Figure  50  shows  a  sim- 
ilar vessel  in  which  two  cultures  may  be  carried  on  simultane- 
ously, or  first  in  one  receiver  and  later  in  the  other,  by  care- 
fully pouring  a  drop  into  it  from  the  first. 

II.  PRESERVATION  OF  A  GIVEN  ANAEROBIC  CULTURE. 

For  keeping  a  pure  culture  upon  solid  media,  all  of  the  ap- 
pliances recommended  above  may  be  used,  and  isolated-colony 
cultures  obtained,  which,  as  a  rule,  are  attended  by  no  incon- 
venience. If  thrust-cultures  are  desired,  other 
methods  must  be  employed,  such  as  the  follow- 
ing four  : 

1.  Use  of  Oil  or  a  Gelatinizing  Plug. — 
The   inoculation -thrust  is  made  as  usual  in  a 
tube  of  agar  or  serum,  but  with  a  glass  needle 
in  preference  to  platinum   (p.   45).       Immedi- 
ately afterward,  a  layer  5  cm.  thick  of  olive-oil 
previously  sterilized  by  boiling,  or  of  sterilized 
2-per-cent   agar  which    has  been   allowed    to 
cool  to  about  40°  C.,  is  poured  into  the  tube, 
with  the  usual  precautions.     The  advantages 
and  disadvantages  of  each  of  these  substances 
are  self-evident. 

2.  Use  of  Entirely  Filled  and  Hermeti- 
cally Sealed   Tubes   (Roux).— A  tube  of  the 

form  shown  in  Fig.  53  a,  is  plugged  with  cot-      FIG.  52.  -  cuitiva- 

r      &  tion     Of     Anaerobic 

ton  at  its  upper  end,  and  the  lower  capillary  species  under  on  or 
portion  is  sealed  by  fusion,  after  which  it  is  Asar- 
sterilized  by  hot  air  at  150°  C.  Sterile  nutrient  gelatin  or 
agar  is  brought  to  the  boiling  point,  and  as  soon  as  it  has 
ceased  boiling  the  opened  capillary  end  of  the  tube  is  dipped 
into  it,  and  the  tube  is  filled  up  to  the  constriction,  by  suction. 


Bacteriological    Technology. 


By  pressing-  a  finger  tightly  over  the  upper  end,  and  quickly 
raising  the  tube  into  an  oblique  position,  the  gelatin  is  pre- 
vented from  running  out  of  the  end,  which  is  at  once  sealed 
in  the  flame.  The  upper  end  is  likewise  sealed  by  fusion  at  the 
constriction  just  above  the  gelatin.  When  the 
contents  are  to  be  inoculated,  one  end  is  opened, 
a  thrust  is  made  with  an  infected  fine  glass 
needle,  and  the  tube  is  again  sealed.  When 
the  developed  culture  is  to  be  examined,  it  is 
best  to  open  the  end  opposite  that  from  which 
the  thrust  was  made,  otherwise  the  colonies 
can  easily  be  forced  from  the  tube  by  the  pres- 
sure of  gas  generated,  during  their  growth. 

3.  Removal  of  Oxygen  by  the  aid  of  Aero- 
bic Bacteria.^  Roux's  method  is  as  follows: 
A  suitable  quantity  of  nutrient  agar  is  boiled 
in  a  cotton-plugged  test-tube,  and  quickly 
cooled  in  cold  water.  Immediately  after  it 
has  become  solid,  it  is  inoculated  by  a  glass 
needle,  after  which  a  small  quantity  of  melted 
but  not  too  hot  nutrient  jelly  is  poured  into 
the  tube,  and,  as  soon  as  it  has  solidified,  a 
couple  of  drops  of  bacillus  subtilis,  or  some 
other  decidedly  aerobic  species,  are  sown  upon 
its  surface.  The  test-tube  is  then  fused  to- 
gether at  top,  and  set  in  the  brood-oven.  B. 
subtilis  grows  rapidly  upon  the  surface,  using- 
up  the  oxygen  above  it,  and  so  preventing  this 
from  reaching  the  agar,  in  which  the  anaerobic 
form  is  consequently  permitted  to  grow  undis- 
turbed. To  obtain  material  for  transfers  from 
this  culture,  the  tube  is  opened  by  breaking  off 
its  bottom,  to  avoid  the  admixture  of  B.  sub- 
tilis. 

Cultures  can  also  be  made  in  the  appa- 
ratus shown  in  Figure  54,  where  the  mixture 
of  the  two  forms  is  entirely  avoided.  The  inner  tube  is  filled 
by  means  of  a  Pasteur  pipette  with  agar-g-elatin,  and  the  space 
about  it,  with  bouillon;  the  former  is  inoculated  with  the 
anaerobic  form,  the  latter,  with  the  aerobic  species,  and  the 
outer  tube  is  sealed  hermetically  at  a. 


FIG.  53.  —  Pipette 
Tubes  for  Cultivating 
Anaerobic  Species 
(Roux). 


Bacteriological    Technology. 


4.  Removal  of  Oxygen  by  Pyrogallic  Acid. — Buckner  has 
recently  recommended  the  following-  method:  The  anaerobic 
form  is  sown  in  a  small  cotton-plugged  test-tube  containing- 
agar,  immediately  after  the  latter  has  been  boiled  and  rapidly 
cooled  down.  This  tube  is  then  supported  on  a  simple  wire 
stand  (Fig.  55)  [or  a  piece  of  spiral  spring]  in  a  larger  test- 
tube,  the  lower  part  of  which  contains  an  alkaline  solution  of 
pyrogallic  acid  (1  g-m.  of  pyrogallic  acid ;  10  cc.  of  10-per-cent 
solution  of  caustic  potash).  The  larger  tube  is  carefully  closed 
by  a  tightly  fitting  rubber  stopper,  which  is  slightly  moist- 


-O, 


FIG.  54. 


FIG.  55. 


FIG.  56. 


FIG.  54.— Method  of  Cultivating  Anaerobic  Bacteria  by  Aid  of  Others  which  Exhaust  the 
Oxygen  (Salomonsen). 

FIG.  55. — Apparatus  for  use  of  Pyrogallic  Acid  (Buchner). 

FIG.  56.— Arrangement  for  Cultivating  Anaerobic  Species  on  Potato  (Roux). 

ened  on  the  sides,  and  the  culture  is  usually  set  in  the  brood- 
oven  at  a  little  over  30°  C.  Buckner  has  satisfied  himself  that 
the  development  of  so  markedly  anaerobic  a  form  as  the 
bacillus  of  malignant  cederna  is  easity  effected  in  this  appara- 
tus, though  a  little  slowly.  Absorption  of  the  oxyg-en  is  hast- 
ened by  forcibly  shaking  the  tube  now  and  then. 

5.  Potato-Cultures  in  a  Vacuum. — Roux  employs  the  ap- 
paratus shown  in  Figure  56  for  this  purpose.  After  the  piece 
of  potato  has  been  sterilized  in  the  steam-cylinder,  and  'the 
condensed  moisture  has  collected  at  the  bottom  of  the  tube, 


Bacteriological    Technology.  93 

below  the  narrowed  portion  6,  the  potato  is  inoculated  in  the 
usual  way,  and  the  test-tube  hermetically  sealed  at  a.  The 
small  lateral  tube  is  then  connected  with  the  air-pump,  the 
exhaustion  is  kept  up  for  several  minutes,  so  that  all  air  may 
escape  from  the  interior  of  the  potato,  and  the  tube  is  sealed 
by  melting-  at  c. 

The  preservation  of  anaerobic  pure  cultures  in  fluid  media 
is  exposed  to  no  difficulties.  The  apparatus  adapted  to  the 
passage  of  hydrogen  (Fig1.  49),  or  to  the  use  of  the  air-pump 
(Figs.  47,  50,  51),  is  employed. 


CHAPTER    IX. 

CULTIVATING     MICRO-ORGANISMS     UNDER     THE     MICRO- 
SCOPE. 

IF  it  is  desired  to  follow  the  development  of  a  microbe  by 
uninterrupted  observation  for  some  hours  or  days,  so  that 
growth,  fission,  the  formation  and  germination  of  its  spores, 
etc.,  may  be  directly  seen,  the  culture  must  be  made  in  a 
"  moist  chamber,"  attention  being  given  to  the  usual  precau- 
tions, such  as  preliminary  sterilization  of  the  several  parts  of 
the  chamber,  rapid  inoculation,  etc.  The  chambers  must  be 
hermetically  sealed  by  melting,  using  cement,  or  in  some  such 
manner,  to  prevent  evaporation  or  infection.  They  must, 
therefore,  be  large  enough  to  contain  a  sufficient  quantity  of 
air  in  addition  to  the  necessary  nutrient  substance.  Investi- 
gations of  this  sort  are  most  frequently  attended  with  some 
difficulty,  especially  for  beginners,  who  can  best  practise  them 
upon  larger  forms,  such  as  yeasts  and  moulds,  passing  from 
these  to  the  spore  formation  and  germination  of  the  large 
species  of  bacillus. 

The  following  rules  are  observed  in  employing  the  various 
moist  chambers,  the  most  important  and  useful  of  which  will 
be  described. 

Cover  glasses  are  cleansed  in  the  usual  manner  with  hydro- 
chloric acid,  followed  by  alcohol  and  finally  by  distilled  water. 
After  careful  drying  they  are  laid  in  a  pair  of  small  glass 
trays  (Fig.  13)  which  are  wrapped  in  paper  and,  with  their  con- 
tents, sterilized  in  the  dry  oven  at  150°  C.  For  further  cer- 
tainty, the  cover-glasses  may  be  drawn  several  times  through 
the  flame,  immediately  before  use.  In  the  same  wray,  the  dif- 
ferent kinds  of  slides  and  chambers  are  sterilized  by  dry  heat, 
wrapped  in  paper,  in  which  they  remain  until  the  moment  of 
use. 

In  preparing  the  moist  chambers  it  is  well  to  use  a  larger 


Bacteriological    Technology.  95 

piece  of  sterilized  paper  to  unwrap  the  slides  upon,  and  to 
cover  them  with  small  bell-glasses,,  leaving-  them  uncovered 
for  only  the  shortest  possible  time.  Cover-glasses  must  be 
treated  with  the  same  care.  The  larger  shallow  glass  trays 
(Fig.  12)  serve  well  as  bell-glasses. 

Several  chambers  are  commonly  employed  at  the  same 
time,  since  some  of  them  are  liable  to  contamination  while 
being  prepared,  even  though  they  are  sowrn  quickly  and  care- 
fully. 

Cell-cultures  are  always  started  from  young  and  strong" 
cultures,  usually  in  the  same  medium  that  is  used  in  the 
moist  chamber.  Care  is  to  be  taken  that  the  germs  are  uni- 
formly distributed,  and  in  proper  number,  one  to  four  in  each 
field  of  the  microscope  being  right.  As  this  depends  upon 
repeated  preliminary  examinations,  three  test-tubes  are  used, 


FIG.  57.— Hollow-ground  Slide  for  use  with  Hanging  Drops. 

each  containing  a  few  grams  of  a  suitable  nutrient  medium. 
A  rather  large  number  of  germs  are  suspended  in  the  first, 
and  the  effort  is  made  by  strong  shaking  to  isolate  them  and 
distribute  them  uniformly.  A  drop  is  then  transferred  by 
means  of  a  platinum  loop  to  the  second  tube,  and  after  thor- 
ough agitation  the  number  of  germs  in  the  new  mixture  is 
determined  under  the  microscope  by  examining  a  drop  of  it  in 
the  sort  of  moist  chamber  to  be  employed  for  the  culture,  and 
with  the  power  to  be  used  for  this.  If  there  are  too  many 
germs,  a  little  fluid  is  added  from  the  third  test-tube,  but  if 
too  few  are  present,  their  number  is  increased  by  adding  a 
drop  from  the  first  tube. 

One  of  the  simplest  and  most  frequently  used  moist  cham- 
bers (in  which  Koch  first  observed  the  growth  and  spore 
formation  of  Bacillus  anthracis  outside  of  the  animal  organ- 
ism, in  1876)  is  the  hollow-ground  slide — a  glass  slip  of  the  form 
and  size  of  an  ordinary  microscope  slide,  with  a  circular  de- 
pression at  the  centre,  about  15  mm.  in  diameter  (Fig.  57). 


96  Bacteriological    Technology. 

After  placing-  a  small  drop  of  culture-fluid,  containing-  the 
microbe  to  be  studied,  on  the  lower  side  of  a  cover-glass,  this 
is  laid  over  the  hollow  and  fastened  to  the  slide  in  an  air-tight 
manner  by  smearing  with  vaselin.  Aside  from  the  general 
rules  given  above,  attention  is  to  be  given  in  the  use  of  this 
cell,  and  in  examination  in  hanging-  drops  to  the  following: 
The  drop  must  be  spread  out  in  as  thin  a  layer  as  possible, 
b'ecause  it  must  usually  be  capable  of  examination  through  its 
entire  depth.  Often  it  is  well  to  place  a  drop  of  sterile  nutri- 
ent fluid  on  the  cover-glass,  and  to  infect  it  at  some  easily 
recognized  point  along-  the  margin  of  the  drop. 

It  is  best  when  the  drop  has  been  placed  on  the  cover,  to 
fix  this  to  the  slide  by  smearintg  a  little  vaselin  around  the  de- 
pression with  a  sterile  glass  rod,  after  which  the  slide  is  lifted 
and  turned  over,  and  pressed  against  the  cover-g-lass  so  that 
the  edges  of  the  latter  are  covered  by  the  vaselin,  while  the 
culture  drop  projects  into  the  hollow.  Instead  of  fluid,  a 
small  quantity  of  nutrient  gelatin  may  be  placed  on  the  lower 
side  of  the  cover-glass,  making  a  miniature  plate-culture. 

The  preparation  is  first  examined  with  a  low  power  to  get 
a  notion  of  the  germs  present,  and  to  find  a  place  where  they 
are  especially  well  distributed,  when  the  slide  is  fastened  on 
the  stage  of  the  microscope  and  the  g-erms  that  have  been 
picked  out  are  found  with  the  higher  power  it  is  wished  to  use 
for  the  continuous  observation.  When  a  hanging  drop  is  to 
be  found  and  brought  into  focus  under  the  microscope,  this  is 
greatly  facilitated  by  the  fact  that  moisture  at  once  con- 
denses upon  the  cover-glass  about  the  drop,  so  as  to  give  the 
cover  a  roughened  appearance.  By  first  finding  this,  and  then 
moving  the  slide  until  it  disappears,  the  margin  of  the  drop 
is  reached.  Cultures  of  this  sort  can  be  kept  on  the  stage  of 
the  microscope  for  days  without  suffering-  harm,  and  the  de- 
velopment of  a  single  germ  can  thus  be  followed,  step  by  step. 

If  it  is  desired  to  hasten  the  process,  or  if  the  temperature 
of  the  room  is  too  cold  for  the  development  of  the  bacteria, 
the  moist  chamber  may  be  placed  on  some  form  of  warm 
stage  (such  as  Schultze's,  Ranvier's,  Israel's,  or  Vig-nal's),  or 
the  thermostat  for  the  microscope,  recommended  by  Panum 
(Nord.  Med.  Arkiv,  1874,  VI.,  No.  7),  may  be  used.  Usually, 
however,  it  is  sufficient  to  place  the  moist  chamber  in  the 
brood-oven,  removing-  it  now  and  then  for  microscopic  exam- 


Bacteriological    Technology.  97 

ination,  care  being-  taken  to  find  the  same  part  of  the  prepar- 
ation each  time.  The  simplest  "  finder  "  for  marking-  a  given 
point  so  that  it  can  quickly  be  found  again  under  the  micro- 
scope, is  that  indicated  by  Hofmann,  viz.,  scratching  a  cross 
on  each  side  of  the  opening  in  the  stage,  one  upright  (+),  the 
other  oblique  ( X ).  When  the  point  to  be  marked  is  centred 
in  the  field,  two  crosses  are  marked  in  ink  on  the  slide  exactly 
over  those  on  the  stage. 

Boettcher's  moist  chamber  (Fig.  58)  consists  of  a  deep 
glass  ring-  fastened  to  a  slide  by  a  cement  which  is  not  injured 
by  heating  to  150°  C.  for  sterilization.  The  experimenter  can 
easily  fasten  the  ring's  by  one  of  the  cements  for  mending- 
glass-ware;  Hansen  recommends  for  the  same  purpose  a  solu- 
tion of  gelatin  in  glacial  acetic  acid,  to  which  finely  pulverized 
potassium  bichromate  is  added  just  before  use. 


YY////////97/////////Y^^^ 

FIG.  58.— Boettcher's  Moist  Chamber. 

A  little  sterilized  water  is  placed  in  the  bottom  of  the  cell, 
and  the  upper  edge  of  the  ring-  is  painted  with  vaselin  to 
fasten  the  cover-glass.  The  chamber  is  used  like  the  hollo w- 
ground  slide,  either  for  examination  in  hang-ing-  drops,  or  for 
the  observation  of  small  plate-cultures  in  g-elatin.  The  latter 
is  the  method  used  by  E.  Chr.  Hansen  in  obtaining-  pure  cul- 
tures of  yeast  for  use  in  breweries,  starting-  from  a  single  cell. 
As  a  rule  he  employs  rather  larg-e  ring-s  (about  25  rnm.  inside 
diameter),  and  round  cover-glasses  of  corresponding-  size.  A 
drop  of  nutrient  gelatin  containing-  the  yeast  is  spread  upon 
the  latter,  with  all  of  the  precautions  indicated  above  ("Med- 
delelser  fra  Carlsberg-  Laboratoriet,"  ii.,  152). 

The  use  of  hanging-  drops  is  attended  with  certain  difficul- 
ties. The  depth  of  the  drop  is  relatively  great,  its  lower  sur- 
face is  not  plane,  and  the  g-erms  easily  chang-e  position  in  it. 
These  difficulties  are  partly  avoided  by  the  use  of  gelatin, 
which,  nevertheless,  can  by  no  means  entirely  replace  fluid 
7 


98  Bacteriological    Technology. 

culture  media  because  of  its  influence  upon  the  movements 
and  mode  and  rapidity  of  growth  of  bacteria.  Bref eld's  film- 
cultures,  described  below,  avoid  these,  but  are  attended  with 
others. 

A  simple  means  of  remedying1  some  of  the  defects  of  the 
hang-ing-  drop  consists  in  covering-  the  lower  surface  of  the 
drop  with  a  fragment  of  cover-glass,  which  adheres  by  capil- 
lary attraction,  insuring-  a  flat  bottom  to  the  fluid.  The  same 
end  is  reached  by  the  use  of  Ranvier's  moist  chamber  (Fig. 
59),  consisting  of  a  slide  with  a  deep  groove  ground  in  the 
centre,  surrounding  a  circular  disk  the  surface  of  which  is 
about  0.1  mm.  lower  than  the  rest  of  the  slide.  The  drop  of 
fluid  is  placed  upon  this  lower  central  part,  vaselin  is  painted 
around  the  groove,  and  the  cover-glass  pressed  down  upon  it. 
In  this  way  the  drop  is  hermetically  inclosed  between  two 


FIG.  59.—  Ranvier's  Moist  Chamber. 

parallel  flat  surfaces,  while  the   groove  serves  as  an  air- 
chamber. 

A  very  expensive  and  fragile,  but  otherwise  excellent 
moist  chamber,  is  the  modification  of  De  Bary's  and  Geissler's 
model  used  by  Brefeld  in  his  study  of  Bacillus  subtilis,  in 
which  he  succeeded  in  following-  the  entire  development  from 
spore  to  vegetative  cells  and  from  these  to  new  spores.  This 
is  a  glass  tube  20  cm.  long-  (Fig.  60),  in  the  middle  of  which  a 
transversely  cylindrical  cell  2  mm.  deep  is  blown,  the  flat  top 
and  bottom  of  which  have  the  thickness  of  a  cover-glass.  To 
prepare  it  for  use,  it  is  thoroughly  cleansed  with  hydrochloric 
acid,  distilled  water,  alcohol,  and  ether,  and  each  end  of  the 
tube  is  plugged  with  cotton.  When  dry  it  is  sterilized  at  150° 
C.  The  microbes  to  be  studied  are  uniformly  distributed  in  a 
suitable  culture  fluid,  until  by  examining  several  drops  spread 
very  thinly,  it  is  found  that  they  are  present  in  the  right 
number  (about  one  to  four  to  each  microscope  field).  One  plug 


Bacteriological    Technology. 


99 


is  now  removed,  the  open  end  is  quickly  flamed  and  immersed 
in  the  fluid,  which  is  drawn  into  the  cell  by  suction  applied  to 
the  other  plugged  end  of  the  tube.  When  the  chamber  is 
quite  full,  the  fluid  is  allowed  to  run  out  again,  the  wet  end  of 
the  tube  is  dried  with  sterilized  filter-paper, 
and  replugged,  both  ends  are  sealed  with  seal- 
ing wax,  and  the  cell  is  ready  for  examination. 
The  inside  of  the  thin-walled  chamber  is  here 
lined  by  a  very  thin  film  of  fluid  containing 
the  germs,  so  thin  that  the  bacteria  do  not 
move  about  in  it.  A  suitable  part  of  the  pre- 
paration is  found,  and  the  chamber  is  fixed 
upon  the  stage  of  the  microscope.  In  search- 
ing through  it,  which  is  done  with  the  lowest 
power  sufficient  for  recognizing  the  germs,  care 
is  taken  not  to  crowd  the  objective  against 
the  thin  and  fragile  wall  of  the  cells,  a  mis- 
hap which  is  rendered  all  the  easier  by  the  fact 
that  the  latter  is  not  perfectly  flat.  It  is  most 
easily  fastened  on  the  stage  by  first  placing  it 
upon  a  thin  and  well-cleansed  slide,  to  which 
the  cylindrical  ends  can  be  attached  by  paraf- 
fin. Admitting  of  the  safe  and  easy  trans- 
mission of  gases,  this  moist  chamber  can  also 
be  used  for  studying  the  development  of  bac- 
teria in  different  gases. 

Other  forms  of  moist  chambers  are  easily 
improvised  out  of  the  material  and  appliances 
to  be  found  in  every  laboratory.  As  it  may 
be  important  at  times  to  work  with  a  very 
large  number  of  cell-cultures  at  the  same  time, 
some  of  these  easily  extemporized  forms  de- 
serve description. 

Buchner  studied  the  germination  of  spores 
of  Bacillus  anthracis  in  a  moist  chamber  such 
as  is  shown  in  Figure  61.  A  very  small  quan- 
tity of  a  pure  culture  containing  only  the  spores  of  this  species 
is  dried  upon  a  cover-glass,  and  a  small  drop  of  culture-fluid 
added.  The  cover  is  then  supported  on  a  slide  at  two  edges, 
by  fragments  of  cover-glass,  the  entire  margin  sealed  with 
some  cement,  and  the  slide  placed  on  a  warm  stage. 


FIG.  60.  —  Geissler- 
chamber  for  Cultiva- 
tion in  a  Film  (Bre- 
feld). 


IOO 


Bacteriological    Technology. 


A  cell,  the  inventor  of  which  is  not  known  to  me,  consists 
of  rather  thick  pasteboard  (No.  16),  cut  into  the  form  shown 
in  Figure  62,  and  carefully  sterilized  by  boiling-  or  in  the  steam 
cylinder,  which  is  best  effected  by  piling  several  pieces  up  be- 
tween a  couple  of  microscope  slides  and  tying  them  firmly 
together.  One  of  the  pieces  of  pasteboard,  while  still  wet  from 
sterilization,  is  laid  on  a  sterile  slide  and  pressed  into  contact 
by  a  glass  rod.  A  cover-glass,  with  the  hanging  drop,  is  laid 
over  the  hole  in  the  pasteboard  and  carefully  pressed  down 
on  it,  after  which  sterile  water  is  added  at  one  edge  until  the 
paper  refuses  to  absorb  more.  In  this  simple  chamber,  the 
paper  serves  both  as  a  source  of  moisture  and  a  means  of  at- 
taching the  cover,  so  that  it  must  be  kept  from  drying  out  as 
long  as  the  chamber  is  in  use ;  consequently,  wrhen  not  under- 
going examination,  cells  of  this  kind,  like  plate-cultures,  need 


FIGS.  61  and  62.— Improvised  Moist  Chambers,    61,  Buchner's  form;  62,  Cardboard  for  Cell. 

to  be  kept  in  a  large  moist  chamber  (cf.  p.  60),  and  from  time 
to  time  the  pasteboard  must  be  resaturated  by  the  addition 
to  the  outer  edge  of  a  little  sterile  water  or  very  dilute  subli- 
mate solution.  For  this  reason  this  form  of  cell  is  not  well 
adapted  to  continuous  observation  under  the  microscope  for 
a  period  of  days,  though  it  may  often  prove  very  useful  as  an 
accessory.  Other  materials  may  also  be  used  for  the  prepa- 
ration of  moist  chambers  of  the  same  sort. 

In  his  studies  of  the  life-history  of  the  higher  fungi,  Brefeld 
has  cultivated  them  under  control  of  the  microscope  by  the 
simple  use  of  slides  upon  which  a  nutrient  fluid  was  spread  in 
a  thin  layer.  These  plate-cultures  in  fluid  are  not  suited  to 
the  study  of  bacteria.  So  far  as  yeasts  are  concerned,  they 
offer  no  especial  advantage  over  cell-cultures,  but  are  attended 
by  certain  disadvantages.  On  the  other  hand,  they  are  of 
the  greatest  importance  for  the  study  of  the  development  of 
higher  fungi,  and  for  this  reason  merit  brief  consideration  here. 


Bacteriological    Technology.  101 

The  solutions  described  on  pages  19  and  20  are  used,  gen- 
erally after  being-  boiled  down  so  that  they  are  not  too  fluid. 
By  means  of  a  glass  rod,  an  elongated  drop  is  placed  on  a 
sterile  slide,  and  enough  of  the  spores  to  be  studied  are  added 
and  distributed  with  a  platinum  needle  so  that  only  one  or 
two  occur  in  each  field  of  the  microscope.  It  is  evident  that 
such  cultures  cannot  be  used  for  prolonged  uninterrupted  ob- 
servation. If  kept  long  upon  the  stage  of  the  microscope, 
they  will  be  ruined  by  drying  out  or  contamination.  Between 
the,  different  examinations,  they  must  be  kept  in  larger  moist 
chambers,  the  air  of  which  is  always  saturated  with  moisture 
(a  common  flat  porcelain  platter  filled  with  water  and  covered 
by  a  bell-glass  dipping  into  the  water),  and  they  can  scarcely 
be  used  successfully  except  in  laboratories  where  the  air  is 
kept  as  free  as  possible,  by  great  care,  from  germs,  and  espe- 
cially moulds ;  but  Bref eld's  works  show  sufficiently  how  far  it 
is  possible  to  utilize  this  method.  Besides  other  advantages 
over  similar  slide-cultures  on  solid  media,  these  admit  of  trans- 
planting the  spores  after  germination  has  begun,  more  favor- 
able conditions  are  given  .the  moulds  for  growth  and  develop- 
ment, and  it  is  possible  to  renew  the  supply  of  food  material 
during  weeks  or  even  months,  as  it  becomes  exhausted,  by 
adding  a  fresh  drop  of  culture-fluid  at  the  margin  every  day 
or  two. 


CHAPTEE    X. 

INOCULATION  OF  ANIMALS. 

WE  come  now  to  a  group  of  experiments  to  which,  in  a 
certain  sense,  all  of  the  preceding-  chapters  point — experi- 
ments calculated  to  give  the  proof  that  a  large  number  of  the 
most  familiar  contagious  diseases  are  induced  by  bacteria, 
and  to  render  possible  a  more  intimate  study  of  the  mutual 
relations  between  these  bacteria  and  the  animal  organism. 
In  this  connection  it  must  be  said  that  conclusive  proof  that 
a  given  infectious  disease  is  due  to  a  specific  bacterian  form, 
is  given  only  when  such  a  form,  well  characterized  morpho- 
logically, chemically,  or  physiologically,  can  always  be  demon- 
strated in  the  organs  by  the  microscope  and  by  cultures,  in 
this  disease  and  only  in  it;  and  when,  further,  these  bacteria, 
after  being  cultivated  pure  for  several  generations  outside  the 
animal  organism,  when  reintroduced  into  an  animal  of  the 
species  they  were  originally  obtained  from,  produce  the  same 
disease  in  this,  and  are  then  demonstrable  in  its  tissues  under 
the  microscope  and  by  pure  cultures. 

Various  kinds  of  animals  have  been  used  for  experiment, 
but  usually  the  smaller  rodents  (rabbits,  Guinea-pigs,  rats, 
and  mice),  or  birds  (fowls  and  pigeons)  are  employed.  It  is 
now  well  known  that  inoculations,  not  of  one  but  of  a  large 
series  of  infectious  diseases,  may  be  effected  upon  one  of  the 
most  accessible,  cheapest,  smallest,  and  most  prolific  of  mam- 
mals— the  white  mouse,  which  is  more  or  less  receptive  for 
splenic  fever,  chicken-cholera,  malignant  oedema,  several  forms 
of  septicaemia,  some  pyaemic  affections,  glanders,  tetanus,  etc., 
etc.  While  the  white  mouse  (an  albino  of  the  house  mouse, 
Mus  musculus)  is  very  slightly  receptive  for  glanders,  ac- 
cording to  Loeffler,  the  European  field  mouse  (Arvicola  ar- 
volis]  is  very  susceptible  to  it  (Loeffler),  as  is  also  the  wood 
mouse  (Mus  sylvaticus)  according  to  Kitt.  On  the  other 


Bacteriological    Technology.  103 

hand,  the  field  mouse  is  not  receptive  for  mouse  septicaemia, 
to  which  the  white  mouse  shows  a  great  susceptibility  (Koch). 
Both  field  and  wood  mice  are  easily  kept  in  captivity  if  fed 
upon  oats  and  moistened  bread.  The  wood  mouse  is  very 
active,  and  its  bite  quite  painful ;  and  it  is  not  advisable  to 
keep  several  individuals  of  either  of  these  mice  in  the  same  jar 
after  inoculation,  because  in  case  one  dies  the  others  devour  it 
at  once.  Where  it  is  only  desired  to  perform  the  most  im- 
portant fundamental  experiments  in  bacterial  infection,  it  is, 
therefore,  usually  sufficient  to  employ  white  mice. 

Keeping  Mice. — After  many  trials,  I  can  highly  recom- 
mend the  use  of  the  cracker-boxes  spoken  of  on  p.  2,  as  cages 
for  mice.  A  large  number  of  holes  about  as  large  as  a  half 
dime  are  made  in  the  cover,  e.g.,  nine  rows,  of  nine  each.  In 
case  the  holes  are  not  large  or  numerous  enough,  evaporation 
from  the  interior  of  the  box  is  checked  too 
much,  and  the  mice  cannot  stand  the  resulting 
humidity.  The  box  is  filled  nearly  half  full  of 
sawdust,  a  little  cotton  is  laid  on  this,  and  the 
cage  is  ready  for  use.  The  mice  are  fed  upon 
white  bread  softened  with  water  and  oats.  The 
sawdust  contributes  largely  to  lessening  offen- 
sive odors,  and,  in  connection  with  the  cotton, 
FIG.  68.-Mousfr.jar.  grain.}luns,  and  the  excrement  of  the  animals, 
forms  a  warm,  soft,  and  dry  mass,  in  which  the  mice  tunnel, 
and  thrive  and  breed  well.  Ten  or  fifteen  adult  mice  can 
easily  be  kept  in  such  a  box,  or  a  still  larger  number  of  young 
ones.  Emptying  the  box  and  renewing  the  sawdust  and  cot- 
ton is  only  necessary  once  a  month  or  even  once  in  two  or 
three  months,  depending  upon  the  number  of  animals  kept  in  it. 
For  isolating  infected  mice,  it  is  best  to  use  common  glass 
pickle-jars,  holding  two  quarts  (Fig.  63).  '  These  jars  are 
readily  disinfected  after  use,  and  the  animals  can  be  easily 
observed  in  them.  Each  is  filled  for  a  third  of  its  depth  with 
sawdust,  and  covered  at  top  by  a  square  piece  of  close  iron 
gauze,  large  and  flexible  enough  so  that  it  can  easily  be  slipped 
over  the  mouth  of  the  jar  and  pinched  into  the  groove  a. 
For  greater  security  a  heavy  object  may  be  laid  upon  the  lid. 
In  this  way  it  is  fastened  securely  enough  so  that  an  animal 
cannot  lift  it  and  escape,  while  it  is  readily  taken  off  and  re- 
placed, and  can  be  disinfected  in  the  flame  without  difficulty. 


IC4  Bacteriological    TccJuwlogy. 

When  white  mice  are  constantly  used  for  inoculation,  they 
are  usually  raised  in  the  laboratory.  Starting  their  propaga- 
tion is  not  always  easy,  since  mice  are  quarrelsome,  and  males 
which  have  not  been  raised  together  often  fight  and  injure 
one  another,  while  they  are  also  apt  to  kill  and  eat  the  young. 
As  a  general  thing,  a  few  pairs  are  taken  for  a  beginning,  and 
kept  apart.  When  a  female  is  near  the  term  of  pregnancy, 
she  is  removed  from  the  male  and  kept  in  one  of  the  mouse- 
jars  just  described,  where  she  remains  with  her  3Toung  ones 
until  their  eyes  are  open,  when  they  are  all  removed  to  one  of 
the  large  cages,  in  which  two  females  and  their  litters  can  be 
safely  kept,  as  a  rule,  provided  the  young  are  of  about  the 
same  age.  The  young  mice  which  grow  up  together  in  this 
way  generally  get  along  so  well  that  it  is  unnecessary  to  re- 
move the  pregnant  females  as  long  as  there  is  room  for  their 
burrows  and  young. 

The  mode  of  inoculating  an  animal,  and  the  point  at  which 
this  is  done,  depend  upon  the  object  in  view.  If  it  is  merely 
desired  to  infect  the  animal,  introducing  the  contagium 
through  a  wound  in  the  skin  suffices  in  many  cases.  Not  in- 
frequent^, however,  it  is  wished  to  imitate  the  natural  infec- 
tion through  the  uninjured  mucous  membrane  of  the  respira- 
tory or  digestive  system.  In  other  cases  it  may  be  necessary 
to  introduce  the  virus  under  the  dura  mater,  by  trephining. 
In  short,  the  most  different  organs  and  parts  of  organs  are 
used  for  the  introduction  of  cultures  of  bacteria,  or  infectious 
material.  Only  the  most  important  of  the  methods  of  inocu- 
lation are  considered  in  the  following  pages. 

In  most  cases  the  inoculation  of  an  animal  is  practically 
painless,  so  that  it  is  anaesthetized  only  when  some  larger 
operation  is  involved,  as  in  trephining  for  inoculation  with 
rabies.  Mice  and  rats  are  placed  under  a  bell-glass  with  a 
wad  of  cotton  moistened  with  ether,  until,  after  a  short  period 
of  excitement,  they  succumb  to  the  anaesthetic.  After  they 
are  removed  from  the  bell-glass,  and  tied,  insensibility  is  easily 
kept  up,  as  they  are  very  tolerant  of  ether.  It  is  otherwise 
with  Guinea-pigs  and  rabbits,  the  latter,  especially,  dying 
easily  when  anaesthetized  with  ether,  or,  particularly,  chloro- 
form. Practice,  however,  enables  the  operator  to  lessen  the 
number  of  deaths  from  this  cause,  and  in  the  Pasteur  Insti- 
tute only  one  to  two  per  cent  of  the  rabbits  die  from  the  an- 


Bacteriological    Technology.  105 

aesthetic,  which  is  administered  by  pouring*  a  teaspoonful  of 
chloroform  over  a  piece  of  absorbent  paper  folded  as  if  for  a 
filter,  which  is  then  held  like  a  cornucopia  over  the  nose  of  the 
animal.  After  a  few  seconds  the  respiratory  movements  stop, 
and  when  they  are  resumed  shortly  afterward,  insensibility  as 
a  rule  is  complete,,  as  soon  as  this  occurs  the  administration 
of  chloroform  is  discontinued. 

Cutaneous  inoculation  is  best  performed  on  mice  by  abrad- 
ing- the  ear  with  a  vaccine  point  dipped  in  the  virus,  although 
it  may  be  effected  anywhere  on  the  body  after  removing-  the 
hair.  Scarification  of  the  skin  followed  by  rubbing-  the  virus 
in,  or  simply  rubbing-  the  latter  into  the  uninjured  skin,  may 
also  be  practised. 

Subcutaneous  inoculation  of  mice  is  best  performed  under 
the  skin  of  the  back,  just  above  the  root  of  the  tail.  The 
mouse  being-  in  a  glass  jar  (Fig.  63),  its  tail  is  seized  between 
the  thumb  and  forefinger  of  the  left  hand  and  drawn  out  over 
the  rim  of  the  glass,  while  the  body  hang-s  into  the  jar,  which 
is  covered  with  a  small  piece  of  board,  held  in  place  by  the 
free  fing-ers  of  the  left  hand,  so  that  there  is  just  room  for  the 
passage  of  the  root  of  the  tail  between  it  and  the  edge  of  the 
jar.  [When  the  usual  cylindrical  jars  of  the  German  labora- 
tories, with  covers  similar  to  those  of  candy -jars,  but  the  top 
replaced  by  coarse  iron  netting  and  weighted,  are  used,  these 
covers  need  only  be  tilted  a  little  at  one  side  to  serve  the  same 
purpose.  If  gray  house-mice  are  used,  they  are  apt  to  be  far 
more  active  than  white  mice,  and  it  is  best  to  quiet  them  by 
holding  a  handkerchief,  moistened  with  a  few  drops  of  ether, 
over  the  cover,  without  carrying  the  etherization  to  the  sur- 
gical point.  In  any  such  operations,  the  long  "  mouse-tongs  " 
of  the  German  dealers  are  a  great  convenience  for  first  seizing 
the  tail  of  an  animal. — W.  T.]  The  hair  just  above  the  tail  is 
clipped  off,  and  by  mean's  of  a  lancet-needle  or  a  pair  of  scis- 
sors a  small  opening  is  made  in  the  skin  and  a  pocket  torn  in 
the  subcutaneous  connective  tissue,  into  which  the  infectious 
material  is  placed.  Solid  bodies  are  introduced  by  means  of 
the  forceps;  very  small  quantities  of  cultures,  etc.,  by  the 
lancet-needle  or  platinum  needle;  and  slightly  greater  quanti- 
ties of  fluid,  by  means  of  capillary  tubes  or  Pasteur  pipettes. 

When  a  considerable  amount  of  fluid  is  to  be  injected  into 
the  subcutaneous  tissue,  a  sterilized  Pravaz  syringe  must  be 


io6  Bacteriological    Technology. 

used.  The  common  Pravaz  syringe  is  not  adapted  to  sterili- 
zation at  high  temperature,  since  the  piston  is  packed  with 
leather,  and  the  tip  cemented  on.  Generally,  a  syringe  is  used 
the  tip  of  which  screws  on  to  a  thread  on  the  glass  cylinder, 
and  with  an  elder-pith  plunger,  so  that  it  may  be  sterilized  by 
either  moist  or  dry  heat  (Straus  and  Collin). 

Material  from  cultures  in  fluid  or  from  vigorous  cultures 
upon  solid  media,  can  easily  be  taken  from  test-tubes  by  the 
pipette  or  platinum  loop.  But  when  the  bacteria  to  be  used 
for  inoculation  grow  in  a  narrow  line  along  the  needle-thrust 
in  a  test-tube,  and  it  is  desirable  to  have  a  relatively  large 
quantity  of  material,  one  should  rather  cut  out  the  entire 
colony  and  use  it  either  with  or  without  melting.  The  process 
is  a  little  different  according  as  the  culture  is  in  gelatin,  agar, 
or  serum. 

A  gelatin  culture  is  dipped  in  warm  water  or  warmed  over 
the  flame  until  the  outer  part  melts  and  the  gelatin  cylinder 
becomes  free  from  the  test-tube,  so  that  it  can  be  shaken  down 
against  the  cotton  plug,  when  the  latter  is  carefully  removed 
and  the  gelatin  allowed  to  fall  into  a  sterilized  watch-glass, 
where,  with  a  sterile  and  slightly  warmed  knife,  all  of  the 
peripheral  part  containing  no  bacteria  is  cut  off  and  removed, 
leaving  only  a  small  prismatic  piece  of  gelatin  containing  the 
colonies. 

Agar-agar,  which  neither  adheres  to  the  glass  so  firmly  as 
gelatin,  nor  melts  at  so  low  a  temperature,  is  removed  in  a 
somewhat  different  manner.  The  test-tube  is  warmed  some- 
what, a  Pasteur  pipette  is  passed  to  the  bottom  of  the  tube, 
between  it  and  the  agar,  and  by  a  strong  puff  through  the 
pipette,  the  entire  culture  can  usually  be  blown  out  of  the 
glass.  If  this  does  not  succeed,  a  warmed  glass  rod,  such  as 
the  handle  of  a  platinum  needle,  is  passed  down  to  the  bottom 
through  the  agar  at  one  side,  so  as  not  to  come  in  contact 
with  the  colonies  of  bacteria.  As  soon  as  it  has  cooled,  it  can 
be  used  to  draw  the  agar  out  by  a  series  of  pushes  and  pulls. 
The  agar  being  removed  into  a  sterilized  watch-glass,  it  is 
treated  just  like  gelatin. 

Serum  is  far  harder  to  manipulate,  because  it  adheres  to 
the  glass  and  cannot  be  removed  by  melting,  so  that  it  is  nec- 
essary to  cut  the  colonies  out  of  the  test-tube  as  well  as  possi- 
ble with  a  long-handled  pointed  knife. 


Bacteriological 


Technology. 


107 


Intravenous  injection  cannot  be  resorted  to  with  mice, 
because  of  their  small  size.  In  the  case  of  larger  animals,  it 
can  generally  be  effected  without  exposing-  the  vein  by  simply 
thrusting-  the  needle  of  the  syringe  through  the  skin  into  the 
cavity  of  the  vessel. 

With  rabbits,  it  is  particularly  easy  to  inject  material  into 
the  veins  of  the  ear  and  leg.  For  the  former,  the  rabbit  is 
wrapped  tightry  and  carefully  in  a  long  towel,  so  that  only  the 
head  projects.  The  ear  is  washed  with  2-per-cent  carbolic 
acid,  partly  to  disinfect  it,  and  partly  because  the  vessels  are 
more  easily  seen  when  the  hair  is  wet.  An  assistant  holds  the 
animal's  head  and  compresses  the  base  of  the  ear,  so  that  the 


FIG.  64.— Injection  of  Culture  into  the  Vein  of  a  Rabbit's  Ear. 

veins  swell,  the  ear  is  seized  between  the  thumb  and  forefinger 
of  the  left  hand  so  that'  it  is  slightly  tense  over  the  side  of  the 
forefinger,  and  the  canula  is  carefully  thrust  through  the  skin 
into  the  vein  (Fig.  64).  The  assistant  now  releases  the  end  of 
the  vein,  the  operator  holds  the  canula  still  in  the  vein  with 
his  forefinger  and  thumb,  and  injects  the  fluid  slowly.  Bleed- 
ing is  easily  stopped  by  brief  compression,  or  by  amadou.  In 
case  the  vein  has  not  been  pierced,  the  fluid  soon  begins  to 
distend  the  surrounding  connective  tissue,  when  it  is  almost 
always  useless  to  withdraw  the  canula  a  little  way  and  make 
another  effort  to  force  it  into  the  vein,  but  another  vein  is 
usually  at  once  chosen  for  injection.  Commonly  the  branch 
of  the  vein  running  along  the  posterior  edge  of  the  ear  is 
selected,  but  there  are  many  other  available  points  on  the  ear. 


io8  Bacteriological    Technology* 

When  the  ear  is  used  for  injection,  there  is  the  further  advan- 
tage that  one  may  be  sure  of  really  intravenous  inoculation 
without  attendant  accidental  inoculation  of  the  wound,  by 
simply  removing-  the  portion  through  which  the  injection  was 
made,  by  a  quick  clip. 

By  connecting-  the  needle-shaped  canula  with  a  larger 
syring-e  by  a  rubber  tube,  large  quantities  of  fluid  may  be  in- 
troduced into  the  veins  of  the  animal,  in  the  same  simple  and 
painless  manner.  As  the  injection  must  be  effected  very 
slowly,  and  in  this  case  requires  a  longer  time,  it  is  best  to 
fasten  the  animal  upon  a  "rabbit-board"  (the  French  model, 
used  in  Marey's  laboratory,  is  far  preferable  to  Czermak's)  so 
that  a  sudden  movement  of  its  head  shall  not  displace  the 
canula. 

For  injection  into  the  leg  veins,  the  rabbit  is  firmly  and 
carefully  wrapped  in  a  long  towel  so  that  only  one  hind  leg  is 
left  free,  the  animal  being  allowed  to  draw  the  other  up  under 
its  body.  An  assistant  sits  with  the  rabbit  upon  his  lap,  one 
hand  extending  the  free  leg  firmly,  while  with  the  other  he 
grasps  and  compresses  the  thigh  a  little  above  the  knee-joint, 
so  as  to  cause  the  veins  to  swell,  his  fore-arm  resting  upon 
the  body  of  the  animal.  One  of  the  subcutaneous  veins  in  the 
lower  joint  of  the  leg  is  found,  the  hair  over  it  is  clipped  off, 
the  skin  washed  with  2-per-cent  carbolic  acid,  and  while  the 
leg  is  further  steadied  by  the  left  hand,  the  canula  is  pushed 
through  the  skin  into  the  vein,  where  it  is  held  by  the  pressure 
of  the  left  thumb. 

Intraperitoneal  inoculation  is  most  conveniently  effected 
with  a  sterilized  Pravaz  syringe.  The  entire  thickness  of  the 
abdominal  wall  is  pinched  up  in  a  longitudinal  fold,  through 
which  the  canula  is  forced  crosswise  so  as  to  emerge  on  the 
other  side.  On  releasing  the  fold,  the  canula  is  carefully  with- 
drawn enough,  so  that  its  point  lies  within  the  body  cavity, 
while  there  is  no  danger  of  injuring  the  intestines. 

In  case  large  quantities  of  fluid  are  to  be  injected  into  the 
peritoneal  cavity,  a  graduated  glass  pipette  may  be  used,  one 
end  of  which  is  plugged  with  cotton,  while  the  other  is  con- 
nected by  rubber  tubing  with  a  canula  similar  to  that  of  the 
Pravaz  syringe.  The  canula  is  inserted  in  the  manner  indi- 
cated, and  the  fluid  blown  in.  The  same  kind  of  inoculation 
can  also  be  effected  with  a  Pasteur  pipette,  the  perforation 


Bacteriological    Technology.  109 

being1  made  with  a  lancet-needle,  beside  which,  as  a  guide,  the 
pipette  is  passed  into  the  abdominal  cavity.  Mice  and  rats 
are  best  etherized  before  inoculation. 

Inoculation  in  the  peritoneal  cavity  is  especially  important 
in  admitting  of  the  introduction  of  very  considerable  quanti- 
ties of  either  fluid  or  solid  substances  into  the  animal.  In  the 
latter  case,  the  body  cavity  must  be  opened  for  a  greater  dis- 
tance along  the  linea  alba.  By  such  laparotomy,  carefully 
performed,  an  entire  organ  from  a  larger  diseased  animal 
may  easily  be  inserted  into  a  smaller  one,  e.g.,  the  heart  and 
kidneys  of  a  rabbit,  into  a  rat. 

Inoculation  into  the  anterior  chamber  of  the  eye  acquired 
especial  importance  in  the  study  of  tuberculosis,  since  by  in- 
serting small  masses  of  tuberculous  material  into  the  eye  Con- 
heim  and  Salomonsen  succeeded  in  inducing1  tubercle  of  the 
iris,  by  which  it  became  possible  to  directly  observe  the  in- 
cubation period  of  miliary  tuberculosis,  and  its  independence 
of  a  preceding  suppurative  process.  It  has  also  recently  been 
used  for  rabies  inoculations. 

The  operation  is  performed  by  fastening  the  rabbit,  on  its 
belly,  usually  on  an  operating  board,  where  its  head  can  be 
perfectly  fixed.  An  assistant  opens  the  eyelids,  most  conveni- 
ently by  sitting  so  as  to  face  the  operator,  grasping-  the  ani- 
mal's head  with  both  hands.  Occasionally  the  nictitating- 
membrane  is  so  large  that  it  must  be  held  back.  With  a  pair 
of  forceps,  a  fold  of  the  conjunctiva  is  seized,  and  with  a  suit- 
able knife  a  cut  2  to  3  mm.  long  is  made  in  the  cornea,  near 
its  margin,  with  the  usual  precautions  observed  in  ophthal- 
mological  operations.  If  the  material  to  be  introduced  is  a 
solid  substance,  it  is  passed  through  the  opening1  by  means  of 
slender  curved  forceps,  usually  with  very  fine  smooth  points 
opening  parallel,  and  by  carefully  stroking  the  cornea  with  a 
DavieFs  spoon,  the  effort  is  made  to  crowd  the  introduced 
material  into  the  bottom  of  the  anterior  chamber.  If  this  is 
not  easily  effected,  it  is  allowed  to  remain  where  it  lies.  Fluids 
are  injected  through  a  (usually)  blunt  and  bent  canula,  in- 
serted into  the  cut.  To  anaesthetize  the  eye  for  the  operation, 
a  2-per-cent  cocaine  solution  is  dropped  into  it,  the  maximum 
action  being  reached  after  fifteen  minutes  (Howe). 

Another  place,  easily  accessible  for  observation,  which  has 
often  been  used  for  inoculations,  is  the  cornea,  in  which  the 


iio  Bacteriological    Technology. 

conditions  are  very  favorable  for  observing*  the  effect  of  bac- 
teria upon  the  connective-tissue  cells.  By  means  of  a  blunt 
needle,  a  large  number  of  pricks  or  scratches  are  made  in  the 
cornea!  tissue,  without  perforating*  it,  and  the  bacteria  are 
gently  rubbed  into  the  wounds;  or  the  needle  may  first  be 
•dipped  into  the  virulent  material, — but  the  first  method  seems 
to  give  surer  results. 

Inoculation  beneath  the  arachnoid,  as  first  used  in  hydro- 
phobia infections  in  Pasteur's  laboratory  (Pasteur  and  Roux), 
is  effected  on  rabbits  and  guinea-pigs  in  the  following-  manner: 
A  cut  is  made  through  skin  and  aponeurosis,  on  the  crest  be- 
tween the  eyes  and  ears,  and  the  edges  of  the  wound  are  held 
apart  by  a  small  tenaculum.  The  trephine  (about  5  to  6  mm. 
in  diameter)  is  applied  behind  the  orbit,  on  one  side  of  the 
middle  line.  When  the  circular  groove  in  the  bone  is  deep 
enough,  the  centre  pin  is  withdrawn  so  that  it  shall  not 
injure  the  dura  mater.  As  a  general  thing,  the  fact  that  the 
bone  is  cut  through,  is  observed  in  time,  but  for  greater  cer- 
tainty it  may  be  seen,  from  time  to  time  whether  the  disk  of 
bone  cannot  be  lifted  out.  When  the  dura  mater  is  exposed, 
it  is  pierced  with  a  needle-shaped  Pravaz  canula,  bent  nearly 
at  right  angles,  which  is  drawn  slightly  toward  the  operator 
to  avoid  injury  to  the  underlying  brain,  and  two  or  three 
drops  of  the  diseased  spinal  cord  infusion  are  injected.  The 
wound  is  cleansed  with  2-per-cent  carbolic  acid  and  closed  by 
a  couple  of  sutures.  In  the  case  of  dogs,  the  skin  is  pushed 
aside  after  the  cut  is  made,  the  temporal  muscle  is  loosened, 
and  the  skull  is  trepanned  in  the  fossa  temporalis,  where  the 
skull  is  thinner,  and  there  is  little  bleeding. 

As  compared  with  all  other  methods  employed  in  the 
study  of  rabies,  inoculation  under  the  dura  mater  has  the 
advantage  of  giving  absolutely  certain  infection,  as  well  as 
the  shortest  and  constant  incubation.  But  according  to  the 
latest  investigations  of  Roux  and  others,  the  far  simpler  inoc- 
ulation in  the  anterior  chamber  of  the  eye  gives  as  certain 
infection  as  the  subdural. 

Pure  virus  is  always  found  in  the  medulla,  etc.,  of  animals 
dead  of  hydrophobia,  which  is  best  suspended  in  sterile  bouil- 
lon or  0.7  per  cent  salt  solution,  and  injected  with  a  Pravaz 
syringe.  The  removal  and  preparation  of  the  material  must 
naturally  be  effected  with  the  greatest  possible  cleanliness,  to 


Bacteriological    TecJinology.  1 1 1 

avoid  accidental  infection.  In  the  Pasteur  Institute  the 
material  for  inoculation  is  prepared  by  snipping-  out  a  small 
piece  of  brain  substance  from  the  bottom  of  the  fourth  ventri- 
cle and  placing  it  in  a  wine  glass  holding  about  150  cm.,  which 
has  been  covered  with  paper  and  sterilized  at  150°  C.  Here  it 
is  rubbed  to  a  soft  paste  by  means  of  a  glass  rod,  and  a  small 
quantity  of  sterile  bouillon  is  gradually  added,  while  it  is  con- 
stantly stirred.  The  glass  is  then  covered  with  paper  and  set 
aside  until  the  coarser  particles  have  fallen  to  the  bottom,  the 
upper,  slightly  turbid,  layer  of  fluid  being  used  for  injection. 

The  medulla  oblongata  retains  its  virulence  unchanged  for 
at  least  a  month  when  kept  in  pure  neutral  glycerin — a  fact 
which  should  be  remembered  whenever  the  occasion  arises  for 
sending  parts  of  the  central  nervous  system  of  man  or  one  of 
the  lower  animals  to  a  distant  laboratory  for  diagnosis. 

Rabies  vaccines  are  prepared  as  follows  (Pasteur  and 
Roux) :  A  rabbit  weighing  two  kilos  and  measuring  about  45  to 
50  cm.  from  the  nose  to  the  root  of  the  tail,  is  inoculated  by 
trepanning  with  "  virus  fixe."  Six  or  seven  days  later  it  shows 
symptoms  of  rabies,  and  dies  on  the  tenth  day.  Before  putre- 
faction sets  in,  the  central  nerve  system  is  removed  as  follows : 
The  skin  is  split  down  the  back  from  nose  to  tail,  dissected  to 
one  side,  the  dorsal  and  cervical  muscles  are  loosened  from 
the  spinal  column  and  cranium,  and  the  spinous  processes  are 
removed  by  bent  shears.  The  nose  is  then  seized  with  a  pair 
of  strong  bone  nippers,  and  so  held  fast  with  the  left  hand, 
while  the  theca  cranii  is  snipped  and  broken  off  by  means  of  a 
pair  of  Liston's  bone  scissors.  Then  the  vertebral  arches  are 
removed  one  by  one,  by  snipping  them  through  on  the  right 
and  left  side,  as  near  the  body  as  possible,  without  injuring 
the  spinal  cord,  and  breaking  them  off.  This  is  more  easily 
described  than  done,  for  the  spinal  cord  is  easily  crushed,  es- 
pecially in  the  cervical  region;  but  with  practice  one  gradu- 
ally learns  to  expose  it  for  its  entire  length  without  injuring 
it  with  the  coarse  instrument  used.  Then  the  cord  is  cut  off 
over  the  cauda  equina,  its  membrane  is  seized  with  forceps 
just  above  the  cut  and  it  is  raised  for  a  distance  of  6  to  7  cm., 
and  all  adhesions  are  cut.  The  loosened  piece  is  cut  off,  a 
sterile  silk  thread  tied  about  one  end,  and  hung  up  (Fig.  65) 
in  a  litre  flask  with  two  openings  previously  plugged  with 
cotton  and  sterilized  at  150°  C.  Enough  caustic  soda  to  cover 


112 


Bacteriological    TecJinology. 


the  bottom  is  dropped  into  the  flask,  the  spinal  cord  is  hung 
from  its  neck,  and  the  whole  set  aside  at  20  to  25°  C.  The 
vaccines  are  prepared  in  the  manner  described  above,  from 
such  pieces  of  spinal  marrow,  hung-  up  and 
dried  for  a  long-er  or  shorter  time. 

Infection  through  the  digestive  tract  is 
effected  by  carefully  ming-ling-  the  contagium 
with  some  food  that  the  animal  is  fond  of, 
which  is  given  it  in  not  too  larg-e  quantity, 
the  animal  being1  previously  kept  without  food 
for  a  day  or  less,  if  necessary.  In  case  of 
mice  and  other  small  animals,  this  is  the  only 
available  method.  In  the  case  of  larger  mam- 
mals (rabbits  and  Guinea  pigs),  simple  feed- 
ing is  also  used  when  large  quantities  of 
solid  material* are  to  be  introduced  into  the  FIG.  es.-Drymg-jar  for 
stomach,  and  it  is  the  best  means  of  inducing  Hydrophobia  v«*ine. 
infection  through  the  alimentary  canal,  because  it  is  in  this 
way  that  natural  infection  by  means  of  food  occurs  as  a  rule. 
Still  it  cannot  always  be  employed,  and  other  means  must 
then  be  resorted  to.  Fluids  (e.g.,  cultures  of  bacteria)  can 
likewise  be  poured  or  injected  through  the  oesophagus,  the 
rabbit  being  wrapped  in  a  towel  with  the  exception  of  its  head, 
and  held  on  the  lap  of  an  assistant.  The  animal  is  made  to 
open  its  mouth  by  slight  pressure  upon  its  cheeks  over  the 
molar  teeth,  and  a  small  wooden  gag  (Fig.  66)  is  inserted  so 
that  its  incisors  rest  in  the  grooves  on  the  upper  and  lower 
surfaces  (b).  Through  the  perforation  in  the  gag,  a  No.  17 
catheter  can  easily  be  introduced  into  the  rabbit's  ventricle. 

Adhering  bits  of  food  show  that 
it  has  been  inserted  right.  In  case 
of  the  Guinea  pig,  a  smaller  cathe- 
ter (usually  a  bulb  catheter)  must 
be  employed,  and  it  requires  to  be 
passed  down  with  especial  caution. 
Solids  are  quickly  introduced 
into  the  crop  of  doves  or  fowls  by 


Fia.  66.— Wooden  Gag  for  Passing  a 
Catheter  into  the  Stomach,  in  Feeding 
Experiments.  6,  Section  of  same. 


opening  the  beak,  and  placing  the  food  in  the  mouth,  as  far  back 
on  the  tongue  as  possible,  when  the  bird  quickly  swallows  it. 
Small  meal  pellets  are  formed  with  cultures  and  other  fluids 
containing  bacteria,  and  these  are  introduced  in  the  same  way. 


Bacteriological    Technology^  113 

The  introduction  of  infectious  fluids  into  the  stomach  and 
intestine  through  the  abdominal  wall,  by  means  of  a  Pravaz 
syringe,  is  more  certain  and  reliable.  If  the  contagium  is  to 
be  introduced  directly  into  the  intestine,  the  abdomen  must  be 
antiseptically  opened,  a  fold  of  the  intestine  found  and  care- 
fully fixed,  and  the  canula  thrust  through  its  wall.  In  this 
way  cultures  of  the  cholera  spirillum  are  injected  into  the 
duodenum  (Nicati  and  Rietsch),  so  that  they  do  not  pass 
through  the  stomach,  and  the  action  of  the  gastric  juice  upon 
the  microbes  is  avoided. 

Pathogenic  bacteria  can  be  brought  in  contact  with  the 
intact  surface  of  the  lungs  by  injection  through  an  opening 
made  in  the  trachea.  For  the  sake  of  entire  certainty  that 
the  virus  is  not  at  the  same  time  introduced  through  the  open 
wound,  this  may  first  be  allowed  to  heal,  leaving  a  small 
tracheal  fistula.  In  the  case  of  large  animals,  Arloing,  Cor- 
nevin,  and  Thomas  have  reached  the  same  end  by  passing  a 
drainage  tube  through  a  metal  canula,  and  injecting  the  de- 
sired material  through  the  former,  in  small  quantities,  so  as  to 
avoid  spasms  of  coughing. 

If  the  natural  means  of  infection  through  the  mucous  mem- 
brane of  the  respiratory  organs  are  to  be  imitated  as  closely 
as  possible,  the  bacteria  must  be  very  finely  and  uniformly 
suspended  in  the  air  inhaled  by  the  animal,  so  as  not  to  cause 
mechanical  irritation.  This  is  best  effected  by  use  of  a  spray  or 
dry  powder.  The  former  method  is  open  to  the  objection  that 
the  spray  contains  not  only  extremely  fine  drops,  but  likewise 
some  which  are  a  little  larger,  so  that  the  mucous  membrane 
of  the  nose,  or  the  entire  animal,  becomes  quite  damp.  To 
avoid  this,  Buckner  joins  the  atomizer  to  a  two-necked  Woulf 
flask  holding  about  three  litres,  where  the  coarser  drops  are 
allowed  to  settle,  while  the  finer,  as  a  nearly  imperceptible 
mist,  are  passed  out  of  one  neck  of  the  flask  through  a  tube 
to  the  inclosed  chamber  where  the  animals  are. 

For  the  second  method,  a  fluid-culture  of  the  bacteria  is 
poured  over  a  rather  large  quantity  of  spores  of  Lycoperdon 
giganteum,  which  are  then  dried  off  over  calcium  chloride, 
and  the  powder  is  blown  into  the  closed  animal  cage  by  the 
bellows  (Buckner).  In  his  experiments  on  the  entrance  of 
spores  of  B.  anthracis  through  the  lungs,  Buckner  used  0.25 
gm.  of  powder  for  a  space  of  three  litres,  and  allowed  the  in- 
8 


ii4  Bacteriological   Technology* 

halation  to  continue  ten  to  fifteen  minutes.  Other  powders 
(carbon,  talc,  etc.)  can  also  be  used  as  a  vehicle  for  cultures  of 
bacteria,  but  the  puff-ball  spores  are  superior  to  all  of  these 
in  being1  very  small,  uniform,  and  of  low  specific  gravity.  In 
case  the  bacteria  are  killed  or  deprived  of  their  virulence  by 
drying-,  this  method,  of  course,  cannot  be  employed. 

In  such  experiments,  the  animals  must  be  untranimeled  so 
that  their  respiratory  movements  are  not  checked  or  altered, 
and  it  is  best,  therefore,  to  let  them  remain  unfastened.  A 
number  of  mice  can  be  placed  together  loose  in  a  tight  tin 
box  with  a.few  breathing  holes  plugged  with  cotton.  If  it  is 
necessary  to  fasten  the  animals  so  as  to  keep  their  heads  in  a 
given  direction,  wire  gauze  can  be  used,  as  it  may  be  bent  and 
shaped  at  will.  A  single  mouse  can  conveniently  be  placed  in 
a  glass  tube  3  to  4  cm.  in  diameter,  with  a  bored  and  split 
cork  in  one  end,  which  serves  to  hold  its  tail. 

When  inhalation  experiments  are  performed  with  bacteria 
that  are  pathogenic  for  man,  the  operator  should  remember 
to  take  sufficient  precautions,  such  as  having  tight  vessels 
and  tubes,  cotton-plugged  air-spaces  about  the  cages  of  the 
animals,  and  long  tubes  for  the  atomizer,  and  the  work  should 
be  done  in  the  open  air. 


CHAPTEE  XL 

CULTURES  FROM  MAN  AND  ANIMALS.     COLLECTING  AND 
PREPARING  PRIMARILY  STERILE   CULTURE-MEDIA. 

IN  attempting-  to  obtain  cultures  of  the  pathogenic  micro- 
organism from  the  blood  or  organs  of  an  animal  which  has 
died  from  an  infectious  disease,  the  rules  given  in  Chapters  I. 
to  III.,  and  V.,  are  to  be  observed,  as  well  as  the  following : 

Since  the  more  or  less  dirty  hair  or  feathers  of  the  animal 
contain  a  possible  source  of  contamination,  it  is  best  to  skin 
or  pluck  the  body  before  opening  it.  All  of  the  parts  covered 
by  hair  may  also  be  washed  with  sublimate  solution,  or  cov- 
ered with  filter-paper  wet  with  sublimate. 

All  instruments  used  for  preparing  the  body  must  be  care- 
fully sterilized.  Since  repeatedly  heating  them  to  a  high  de- 
gree in  the  flame  w^orks  destructively  upon  knives,  scissors, 
and  forceps,  it  is  best  to  have  two  sets  of  these  instruments; 
one  intended  for  the  coarser  part  of  the  work,  sterilized  by 
flaming,  and  commonly  used  so  hot  as  to  cause  the  flesh  to 
hiss  while  the  cut  is  being  made;  the  other  for  finer  opera- 
tions, first  sterilized  at  150°  C.,  in  a  metal  case  or  wrapped  in 
paper,  and  merely  hastily  flamed  just  before  use. 

In  addition  to  the  gas  or  spirit  flame,  a  dish  of  0.1  per  cent 
sublimate  solution,  and  of  5-per-cent  carbolic  acid,  must  be 
kept  ready  for  the  disinfection  of  hands  and  instruments.  To 
avoid  contamination  from  contact,  the  sterilized  instruments 
are  laid  upon  glass  supports  (Fig.  18)  and  covered  with  a  bell- 
glass,  or  dropped  into  a  short  cylindrical  vessel  with  their 
points  projecting  out  at  top. 

While  exposing  the  organs  from  which  material  is  to  be 
taken  for  cultures,  use  is  made  of  strongly  heated  instruments, 
which,  as  far  as  possible,  are  kept  from  touching  the  organs. 
No  definite  rules  can  be  given,  further  than  this : 

If  a  culture  is  to  be  started  from  fluid  contained  in  a  cavity, 


1 1 6  Bacteriological    Technology. 

it  is  best  to  singe  the  surface  over,  before  piercing1  the  wall. 
For  example,  if  a  collection  of  fluid  in  the  pleural  cavity  is  to 
be  used,  after  exposing  the  ribs  and  intercostal  muscles  in  the 
usual  way,  a  part  of  one  of  the  intercostal  spaces  is  scorched 
by  means  of  a  glass  rod  heated  in  the  flame,  and  then,  with  a 
glowed  needle  or  strongly  heated  small  knife,  the  pleural  cav- 
it}^  is  opened,  and  the  small  gaping  wound  made  for  inserting 
the  inoculation-needle,  capillary  tube,  or  pipette,  according  to 
the  quantity  of  fluid  needed.  In  the  same  manner,  blood  is 
obtained  from  one  of  the  anterior  chambers  of  the  heart.  After 
cauterizing  the  surface,  as  a  rule  the  capillary  tube  or  pipette 
may  be  thrust  through  the  thin  wall,  without  the  previous 
use  of  a  knife,  which  only  becomes  necessary  when  a  thin  and 
flexible  platinum  needle  is  to  be  inserted. 

To  obtain  material  from  the  substance  of  a  solid  organ, 
one  of  the  thicker  and  more  rigid  needles  is  used.  The  organ 
may  be  snipped  in  two  with  scissors  carefully  flamed  or  even 
glowed,  and  the  needle  thrust  into  its  substance  from  the  un- 
contaminated  cut  surface,  or  it  may  be  torn  open  with  sterile 
fingers  or  forceps,  and  in  this  way  a  surface  exposed  which 
has  not  been  touched  at  all  by  the  instruments.  [Loeffler 
advises  that  when  parts  of  cut  organs  are  to  be  broken  in  this 
way,  the  surface  be  first  disinfected  by  dropping  the  whole 
piece  into  0.1  per  cent  sublimate,  and  moving  it  about  in  this 
for  a  minute  or  two].  By  using  flamed,  but  not  too  strongly 
heated,  curved  scissors,  small  pieces  of  the  organ  may  be 
snipped  out  and  placed  on  or  in  the  culture  medium,  at  least 
after  being  crushed  between  two  sterilized  glass  slides.  This 
method  is  recommended  by  Koch  in  sowing  from  tubercle. 

Since  the  white  mouse  is  the  animal  presumably  used  in 
the  elementary  inoculation  experiments,  the  following  descrip- 
tion is  given  of  the  best  means  of  obtaining  cultures  from  its 
heart  blood.  As  soon  as  the  external  examination  is  com- 
plete— especially  about  the  inoculation  wound — the  body  is 
extended  upon  its  back  by  a  shawl  pin  through  each  leg,  on  a 
small  board, -which  it  is  well  to  first  cover  with  a  sheet  of  par- 
affined paper  over  which  a  couple  of  sheets  of  filter-paper  are 
laid  to  absorb  blood,  etc.  The  head  is  finally  fixed  by  means 
of  a  fifth  pin  through  the  nose.  The  skin  is  dissected  away 
from  the  ventral  surface  as  far  as  possible,  and  entirely  cut 
off,  the  occasion  being  taken  to  examine  the  axillary  and  in- 


Bacteriological    Technology.  1 1 7 

guinal  lymphatics,  and,  if  necessary,  to  obtain  blood  from  the 
axillary  vessels  for  microscopic  investigation.  Seizing-  the 
prominent  ensiform  process  with  a  pair  of  sterilized  forceps, 
it  is  ]  if  ted  strongly,  and  ~by  means  of  flamed  sharp-pointed 
scissors,  one  end  of  which  is  inserted  under  the  ribs,  a  suffi- 
cient part  of  the  thoracic  wall  is  removed  to  expose  the  peri- 
cardium. In  this  way  the  intestines  are  not  approached,  and 
the  instruments  are  not  exposed  to  infection  from  this  dan- 
gerous source.  Care  is  taken  to  avoid  touching  the  anterior 
surface  of  the  heart  with  the  scissors.  With  two  pairs  of 
small  flamed  forceps  the  pericardium  is  now  torn  open,  and  a 
small  cut  is  made  into  the  exposed  heart.  Usually  sufficient 
blood  to  wet  the  platinum  needle  at  once  bubbles  out  of  the 
wound;  otherwise  the  needle  is  pushed  through  the  opening, 
and  the  culture  started  from  the  fluid  obtained.  The  dissec- 
tion is  now  completed  in  the  usual  way,  but  with  especial  care 
and  cleanliness  so  as  to  avoid  rendering  it  difficult  or  impossi- 
ble to  make  cultures  from  other  organs  in  which  morbid 
changes  may  subsequently  be  discovered.  When  the  dissec- 
tion is  finished,  and  the  necessary  preparations  have  been 
saved,  the  cadaver  is  wrapped  in  the  filter-paper  it  lies  on,  and 
burned  up. 

The  manner  of  making  the  dissection  naturally  varies  with 
the  organ  chosen  for  starting  a  culture,  the  rule  being  to  pro- 
ceed directly  to  the  organ  from  which  the  chief  culture  is  to 
be  made.  If,  for  instance,  it  is  especially  important  to  ob- 
tain a  culture  from  the  pulp  of  the  spleen,  the  mouse  is  laid  on 
its  side  with  the  spleen  up,  so  that  it  can  be  exposed  without 
disturbing  the  other  organs  too  much. 

The  directions  and  examples  given  here  may  serve  as  a 
sufficient  introduction  to  the  methods  of  obtaining  cultures 
from  the  dead  body,  as  well  as  to  the  collection  and  similar 
use  of  fluids  and  tissues  from  the  living  body,  where,  however, 
consideration  for  the  patient  may  necessitate  many  modifica- 
tions :  e.g.,  scarification  with  the  heated  glass  rod  must  be  re- 
placed by  a  thorough  cleansing  with  disinfectants,  the  Pasteur 
pipette  must  give  place  to  the  sterilized  Pravaz  syringe,  etc. 

Few  appliances  are  needled  for  starting  such  cultures  with 
all  requisite  precautions,  even  at  the  sick-bed  or  operating 
table,  or  for  collecting  the  morbid  products  in  a  state  of  com- 
plete purity,  for  further  elaboration  in  the  laboratory.  Even 


1 1 8  Bacteriological    TccJinology. 

when  much  is  aimed  at,  practically  all  that  is  needed  is  the 
usual  disinfectants  (carbolic  acid  and  corrosive  sublimate) ;  a 
little  sterile  absorbent  cotton  and  filter-paper;  scissors,  knife, 
and  forceps,  wrapped  in  paper  and  sterilized  at  140°  C. ;  steril- 
ized Pravaz  (or  Straus)  syringe;  alcohol  lamp;  platinum  wire; 
capillary  tubes;  Pasteur  pipettes  and  others  with  a  capillary 
constriction  (Fig-.  53,  a) ;  small  test-tubes  containing-  various 
culture  media,  some  of  them  for  the  culture  of  anaerobic 
forms,  e.g.,  with  a  considerable  depth  of  nutrient  gelatin; 
labels ;  and  cover-glasses.  Some  of  these  can  be  prepared  on 
short  notice  in  the  sick-room,  some  are  only  needed  for  excep- 
tional use,  while  the  rest  should  always  be  kept  "in  the  hospital 
wards  to  prevent  the  waste  of  much  valuable  material. 

The  collection  of  primarily  sterile  culture-media,  which 
should  really  have  been  con- 
sidered in  the  third  chapter, 
is  treated  here,  because  the 
precautions  to  be  observed 
are  exactly  the  same  as  in 
obtaining  cultures  from  the 
dead  body,  the  collecting  ap- 
paratus only  being  different. 
Usually  it  is  desired  to  col- 
lect the  sterile  fluids  (blood, 
urine,  milk,  etc.)  either  in 

rather  large  vessels  from  which  they  can  be  transferred  to  a 
number  of  smaller  culture-glasses,  or  they  are  at  once  drawn 
into  the  latter. 

In  the  first  case,  the  so-called  Pasteur  wash-bottle  (Fig.  67) 
is  best  used.  As  the  Figure  shows,  this  is  really  only  a  very 
large  Pasteur  pipette,  with  a  reservoir  of  peculiar  form. 
When  the  capillary  point  b  has  been  sealed  by  fusion,  and  a 
cotton  plug  inserted  at  a,  it  is  sterilized  at  150°  C.  Just  be- 
fore use,  the  tip  ft  is  broken  off,  and  the  entire  capillary  part 
is  flamed.  Observing  the  precautions  indicated  above,  the 
point  is  inserted,  and  the  receiver  sucked  full,  after  which  the 
point  b  is  again  fused.  As  soon  as  possible  afterward,  the 
sterile  fluid  is  transferred  to  smaller  culture-vessels,  by  blow- 
ing through  a,  after  once  more  breaking  the  point  off  and  in- 
verting the  reservoir.  The  test-tubes  or  Chamberland  flasks 
which  have  been  filled  in  this  way  are  kept  on  probation  at 


Bacteriological    Technology*  119 

30°  to  40°  C.,  for  several  clays  before  being  used.  In  this  way 
it  is  possible,  for  instance,  to  collect  large  quantities  of  sterile 
urine  from  the  newly  slaughtered  animals  at  any  slaughter- 
house, by  passing  the  capillary  tip  of  the  flask  through  the 
wall  of  the  bladder,  after  the  abdominal  cavity  is  opened. 

Occasionally  it  may  be  convenient  to  collect  the  fluid  at 
once  in  smaller  culture  vessels.  For  example,  if  sterile  blood 
is  to  be  taken  from  the  heart  of  a  newly  killed  animal,  it  would 
coagulate  in  the  larger  vessel  before  it  could  be  transferred 
to  the  smaller  ones.  In  such  cases,  Pasteur  pipettes  may  be 
used,  not  closed  with  simple  cotton  plugs,  as  is  usual,  but  with 
rubber  tubes  plugged  with  cotton  (Fig.  68),  which  can  be  re- 
moved and  again  replaced  with  far  less  danger  of  contamina- 
tion when  the  contents  of  the  tube  are  to  be  inoculated.  The 
tip  of  the  pipette  is  opened,  flamed,  and  inserted  into  the  heart 
of  a  newly  killed  healthy  animal,  the  tube  sucked  full  of  blood, 
once  more  hermetically  sealed  below,  and  set  aside  some  time 


FIG.  68.— Pasteur  Pipette  with  Rubber  Cap  Plugged  with  Cotton. 

for  observation,  after  which,  if  free  from  germs,  it  may  be 
used  directly  as  a  culture  vessel.  In  the  same  manner,  fresh 
white  of  egg,  containing  no  germs,  can  be  collected  from  hens' 
eggs  (cf.  p.  33). 

The  collection  of  sterile  fluids  from  living  men  or  other 
animals  is  not  always  as  easy  or  certain  as  in  these  cases, 
though  it  is  by  no  means  impossible. 

For  the  means  of  obtaining  sterile  blood  by  phlebotomy, 
see  p.  31.  Milk  can  be  drawn  into  sterile  test-tubes,  after  a 
very  thorough  cleansing  of  the  surface  of  udder  and  teats; 
but  one  should  expect  to  find  a  portion  of  the  tubes  accident- 
ally contaminated.  Urine  can  be  obtained  in  a  sterile  condi- 
tion either  by  passing  it  directly  into  sterile  vessels  or  draw- 
ing it  with  a  catheter,  after  a  careful  disinfection  of  the 
urethra  by  washing  it  out  several  times. 

Now  and  then  very  good  opportunities  occur  for  the  collec- 
tion of  a  large  quantity  of  sterile  culture  fluid  from  living 
patients.  Dropsical  and  other  similar  fluids  can  be  collected 
free  from  germs  without  great  difficulty.  For  this  purpose, 


1 2O  Bacteriological    Technology. 

a  small  Southey  trocar  is  used,  and  the  fluid  is  collected  in  a 
large  flask  furnished  with  a  rubber  stopper  bored  with  two 
holes,  one  of  which  is  plugged  with  cotton,  while  the  other 
admits  a  short  glass  tube  which  is  joined  by  rubber  tubing  to 
the  trocar.  The  instrument  and  glass  ware  are  sterilized  at 
140°  C.,  while  the  rubber  tubing  and  stopper  are  steamed  be- 
fore use. 

The  rules  for  preparing  primarily  sterile  infusions  are  im- 
plied in  the  preceding.  Meat  infusion,  for  instance,  is  obtained 
by  cutting  a  piece  of  muscle  from  a  recently  slaughtered  ani- 
mal, as  rapidly  as  possible,  and  with  all  care,  and  dropping  it 
into  sterilized  water.  Vegetable  infusions  are  generally  more 
easily  prepared,  if  rather  large  organs,  such  as  fleshy  roots 
and  tubers,  are  employed.  The  surface  of  these  is  cleansed 
with  sublimate  solution,  after  which  prismatic  pieces  are  rap- 
idly cut  from  the  interior  with  a  knife  that  has  been  heated 
in  the  flame  so  that  it  hisses  with  each  cut.  From  the  nature 
of  the  case,  rules  for  each  particular  instance  cannot  be  given, 
often  it  is  necessary  to  specially  prepare  the  substance,  e.g., 
in  forming  the  primarily  sterile  infusion  of  jequirity  as  de- 
scribed by  Salomonsen  and  Christmas  ("Hosp.  Ticl.,'1  1884). 

It  is  always  to  be  expected  that  a  certain  number  of  the 
fluids  and  infusions  prepared  in  the  ways  indicated  above  have 
become  accidentally  contaminated  in  the  process  of  prepara- 
tion; hence  they  must  never  be  made  use  of  until  they  have 
been  kept  several  days  in  the  thermostat  at  about  35°  C.,  re- 
maining completely  clear  and  unchanged. 


CHAPTER  XII. 

DISINFECTION  EXPERIMENTS. 

WHEN  it  is  desired  to  learn  whether  a  disinfectant  is  capa- 
ble of  destroying-  a  given  contagium,  the  most  natural  way 
is  to  expose  this  particular  germ,  if  it  is  known,  or  if  not,  sub- 
stances which  carry  it,  to  the  influence  of  the  disinfectant, 
after  which  it  is  used  for  the  inoculation  of  a  suitable  organ- 
ism. But  the  difficulties  in  the  way  of  such  a  test  of  disinfect- 
ants are  evident.  Often  the  contagium  is  one  to  which  our 
experimental  animals  are  not  susceptible,  and  even  when  in- 
oculations are  possible,  they  are  often  too  complicated  to  be 
feasible  on  a  large  scale.  Hence,  long  before  pathogenic  bac- 
teria could  be  isolated  in  cultures,  putrefactive  bacteria  and 
other  micro-organisms  were  employed  as  substitutes  for  the 
real  contagia,  in  investigations  concerning  the  efficacy  of  disin- 
fectants, because  of  the  resemblance  of  pathological  processes 
to  those  of  putrefaction  and  fermentation. 

To-day,  with  greater  reason,  non -pathogenic  bacteria  can 
be  employed  as  tests  of  the  relative  disinfecting  power  of 
various  substances ;  for  we  now  know  that  a  large  part  of  the 
contagia  really  are  bacteria,  and  a  better  knowledge  of  the 
natural  history  of  the  latter  has  rendered  possible  the  avoid- 
ance of  numerous  errors  pertaining  to  earlier  investigations 
and  results.  For  instance,  it  is  now  known  that  a  motile 
species  is  not  to  be  regarded  as  dead  because  its  motions 
cease;  and  the  extremely  different  resisting  power  of  different 
species,  and  the  surprising  vitality  of  spores,  are  now  recog- 
nized, as  well  as  the  fact  that  conclusions  cannot  be  drawn 
for  bacteria  in  general,  and  hence  for  all  contagia,  from  what 
is  learned  of  casual  mixtures  of  bacteria.  E.g.,  the  bacillus 
of  typhoid  fever  is  very  resistant  toward  carbolic  acid,  a  cir- 
cumstance which,  according  to  Chantemesse  and  Vidal,  facil- 
itates their  recognition  in  mixtures  of  bacteria,  since  they  are 


122  Bacteriological    Technology. 

capable  of  developing  in  nutrient  gelatin  which  contains  0.2  per 
cent  of  carbolic  acid.  On  the  other  hand,  substances  are  to 
be  found  which  are  poisonous  to  a  single  species,  while  they 
are  quite  harmless  for  others:  e.g.,  iodoform,  on  the  anti- 
bacterial influence  of  which,  blind  reliance  was  placed  before 
the  investigations  of  Rovsing  and  Heyn.  But  while  this  sub- 
stance is  not  fatal  to  the  greater  number  of  bacteria,  such  as 
Micrococcus  pyogenes  aureus,  the  tubercle  bacillus  (Rovsing) 
and  a  number  of  other  pathogenic  forms,  either  within  the 
animal  organism  or  in  cultures,  it  is  extraordinarily  destruc- 
tive to  the  cholera  spirillum  (Buckner). 

Koch's  directions  for  experiments  with  disinfectants  will 
be  followed  here  very  closely,  since  he  has  recentty  stated  the 
problem  clearly,  and  given  the  simplest  and  best  means  of 
solving  it.  It  is  also  well  known  that  the  researches  of  Koch, 
Gaffky,  and  Loeffler  concerning  many  points  have  caused  an 
entire  revolution  in  the  methods  of  disinfection. 

The  rules  to  be  followed  in  testing  the  germicidal  value  of 
a  disinfectant,  are,  briefly,  the  following : 

Pure  cultures  of  well-known  bacteria  are  used  as  reagents, 
not  mixtures  of  unknown  composition.  For  a  complete  knowl- 
edge of  the  rank  of  the  disinfectant,  these  pure  cultures  should 
represent  the  several  groups  of  micro-organisms  which  are 
active  in  disease  and  fermentation,  i.e.,  moulds,  yeasts,  and 
bacteria.  Bacteria  which  do  not  form  spores  should  also  be 
represented,  as  well  as  the  most  resistant  bacilli  (e.g.,  the 
hay  bacillus,  Chapter  IV.).  But  if  it  is  only  desired  to  learn 
whether  a  given  substance  is  capable  of  destroying  all  organic 
life,  and  consequently  all  living  contagia,  it  is  sufficient  to  use 
bacillus  spores  alone,  since  these  are  the  most  resistant  living 
beings  known  at  the  present  time. 

The  forms  chosen  as  reagents  oug'ht  to  be  easily  recog- 
nizable by  macroscopic  characters;  consequently  chromogenic 
species  are  preferable,  such  as  the  black  Aspergillus  niger, 
the  pink  yeast  ["  Saccharomyces  glutinis  "],  the  blood-red  Mi- 
crococcus  prodigiosus,  etc.,  etc.  The  persistence  or  absence 
of  ability  to  develop  after  exposure  to  the  action  of  the  disin- 
fectant is  taken  as  evidence  of  their  life  or  death. 

According  to  the  nature  of  the  disinfectant,  and  the  cir- 
cumstances under  which  it  is  tested,  the  cultures  employed  as 
reagents  are  used  in  various  ways.  Sometimes  they  are  not 


Bacteriological    Technology.  123 

prepared  in  any  way;  e.g.,  a  tube  or  flask  with  the  contained 
bacteria  and  culture  medium  can  be  exposed  to  a  higii  tem- 
perature for  a  given  time,  and  the  power  of  its  contents  to 
develop  tested  by  inoculation  in  a  fresh  tube;  or  a  drop  of  a 
pure  culture  can  be  distributed  through  a  disinfecting-  fluid 
and  the  mixture  sown  upon  a  suitable  culture  medium. 

But  it  is  generally  more  convenient  to  use  the  cultures 
dried  upon  a  solid  substance,  in  which  condition  they  are 
more  easily  preserved,  transported,  and  made  use  of.  They 
may  be  employed  in  either  of  the  following  ways : 

The  pure  zoogloea  is  sliced  off  from  a  potato  culture  by 
means  of  a  flamed  knife,  with  as  little  of  the  tissue  of  the 
potato  as  possible,  and  laid  away  to  dry  in  a  sterilized  glass 
tray  which  is  wrapped  in  a  double  layer  of  filter-paper  instead 
of  being  covered  by  the  lid.  This  prevents  the  access  of  dust, 
while  it  permits  evaporation  to  go  on  rapidly  enough  to  com- 
pletely dry  the  culture  in  a  couple  of  days.  The  dried  cultures 
are  kept  in  sterilized  test-tubes  plugged  with  cotton,  until  they 
are  to  be  used. 

Small  pieces  of  filter-paper  sterilized  at  150°  C.  are  rubbed 
upon  the  potato  culture  to  be  used,  by  the  aid  of  a  sterilized 
glass  rod,  and  dried  and  preserved  as  in  the  preceding  case, 
after  being  cut  into  narrow  strips  with  a  pair  of  scissors. 
These  strips  are  subsequently  cut  into  smaller  square  pieces 
when  they  are  to  be  used.  In  case  of  fluid  cultures,  the  paper 
may  be  saturated  with  them,  and  dried,  but  it  is  better  to  use 
the  next  method. 

White  silk  thread  is  cut  into  pieces  8  to  10  mm.  long,  which 
are  sterilized  at  150°  C.  in  test-tubes  plugged  with  cotton. 
Though  they  may  be  rubbed  upon  solid  culture,  as  in  the  last 
case,  these  are  especially  suited  to  fluid  cultures,  into  which  a 
large  number  of  them  are  dropped.  After  being  well  shaken 
about  here  for  some  time,  they  are  taken  out  and  dried  as 
above,  care  being  taken  to  lay  them  in  the  glass  well  sepa- 
rated, since,  especially  when  taken  from  liquefied  gelatin  cul- 
tures, they  are  very  apt  to  cling  together.  They  are  preserved 
as  in  the  preceding  cases. 

Frequent  use  is  made  of  the  spores  of  Bacillus  anthracis 
and  B.  subtilis,  in  disinfection  experiments.  To  be  sure  of 
abundant  formation  of  spores  in  the  former,  it  must  be  sown 
on  the  surface  of  nutrient  agar,  and  kept  in  the  brood-oven  at 


1 24  Bacteriological    Technology. 

30°  to  37°  C.,  for  a  week  or  longer.  When  it  is  found  by  mi- 
croscopic observation  that  fully  developed  normal  spores  are 
abundant,  and  the  vegetative  filaments  beg-in  to  disappear,  1 
to  2  cm.  of  sterilized  water  is  added  to  the  tube  containing  the 
spore  bearing  culture,  which  is  distributed  through  it  by  ener- 
getic shaking.  The  pieces  of  sterile  silk  are  dropped  into  the 
turbid  fluid,  and  preserved  in  the  manner  indicated.  The 
hay  bacillus  is  always  certain  to  form  its  spores  in  a  fluid 
medium  such  as  bouillon.  It  is  sown  in  a  conical  flask  con- 
taining bouillon  to  a  depth  of  2  cm.,  and  kept  at  30°  to  37°  C. 
A  day  or  two  later  it  has  developed  a  firm  spore-bearing  mem- 
brane upon  the  surface  of  the  turbid  liquid.  After  some  time 
the  culture  becomes  exhausted  and  loses  its  turbidity,  the  spores 
sinking  to  the  bottom  as  a  white  powder.  Three-fourths  of 
the  supernatant  fluid  is  poured  off,  the  precipitate  of  spores 
is  shaken  up  in  the  remainder,  and  silk  is  saturated  with  it. 

If  spores  are  wanted  which  have  even  more  vitality  than 
those  of  these  two  species,  they  may  be  obtained  from  garden- 
earth,  etc. 

The  spores  of  moulds  are  best  prepared  in  a  somewhat  dif- 
ferent manner.  The  mould  is  sown  upon  gelatinized  beer-wort 
in  a  pair  of  small  glass  trays  (Fig.  13).  When  it  has  covered 
the  surface  and  is  in  fruit,  the  entire  culture  c'an  be  stripped 
off  in  one  piece,  when  it  is  laid  upon  a  piece  of  filter-paper  in 
which  it  is  loosely  wrapped  to  prevent  the  scattering  of  the 
spores,  and  when  dry  it  is  clipped  into  strips. 

Garden  earth  has  also,  by  the  advice  of  Koch,  frequently 
been  used  as  a  bacteriological  reagent  in  disinfection  experi- 
ments. In  this  case  the  rule  that  only  pure  cultures  are  to 
be  used  is  set  aside,  and  in  this  fact  lies  the  disadvantage  of 
using  earth,  which  contains  different  bacilli  in  different  places, 
so  that  it  is  impossible  to  draw  general  conclusions  from  the 
results  obtained  with  a  single  sample  of  earth.  On  the  other 
hand,  it  has  the  advantage  of  being  quickly  prepared  any- 
where with  ease;  but  in  comparative  mvestigations  it  is  neces- 
sary to  use  small  portions  of  the  same  bit  of  earth  for  the 
entire  series.  Pure  cultures  of  the  most  resistant  earth 
bacilli  can  easily  be  obtained  by  suspending  a  rather  large 
quantity  of  dirt  in  bouillon,  which  is  afterward  boiled  in  a 
cotton-plugged  flask  for  15  to  30  minutes,  set  aside  at  30°  to  40° 
C.  for  a  couple  of  days,  and  then  plated  out  in  agar-gelatin. 


Bacteriological   Technology.  1 2  5 

It  must  be  remembered  that  these  reagents,  excepting1, 
perhaps,  the  bacillus  spores,  cannot  be  kept  in  a  useful  state 
for  an  unlimited  time,  but  that  they  generally  die  after  a  longer 
or  shorter  time.  It  is,  therefore,  necessary  to  ascertain  their 
ability  to  develop  before  using  them  in  case  they  are  rather 
old.  It  is  safest  to  use  freshly  prepared  material. 

The  general  method  of  carrying  out  an  investigation  is  as 
follows :  1,  exposing  the  bacteria  to  the  action  of  the  disinfec- 
tant; 2,  removing  the  latter;  3,  sowing  or  inoculating  the 
exposed  bacteria  on  a  suitable  culture  medium  or  animal;  4, 
sowing  on  inoculating  control  material  at  the  same  time ;  5, 
observing  the  result  of  the  cultures  or  inoculations,  not  merely 
as  to  whether  or  not  they  grow,  but  also  rapidity  of  growth, 
virulence,  etc.,  etc. 

When  it  has  been  shown  by  such  experiments  that  a  given 
disinfectant  really  destroys  one  of  the  pathogenic  bacteria 
quickly  and  surely,  it  by  no  means  follows  that  the  substance 
is  practically  useful  for  the  destruction  of  this  contagium. 
Before  a  disinfectant  can  be  recommended,  it  is  particularly 
necessary  to  show  that  it  not  only  kills  the  germs  on  our  silk 
threads,  bits  of  papers,  and  potatoes,  but  is  also  capable  of 
attacking  them  as  they  occur  in  nature — in  clothing,  excre- 
ment, sputum,  etc.  The  effectiveness  of  the  most  powerful 
disinfecting  agents  may  under  such  circumstances  be  neutral- 
ized by  chemical  changes  or  the  impermeability  of  the  sub- 
stance which  harbors  the  contagium,  which,  for  example,  pre- 
vents the  successful  employment  of  the  corrosive  sublimate 
solution  for  disinfecting  vaults  and  tuberculous  sputum.  On 
the  other  hand,  contagia  may  occur  in  nature  under  circum- 
stances more  favorable  for  their  destruction  by  a  given  disin- 
fectant than  is  the  case  when  pure  cultures  of  them  are  ex- 
posed to  its  action.  [Laplace's  solution  of  sublimate  in  crude 
hydrochloric  acid,  diluted  for  use  to  the  standard  ratio  of  1 : 
1,000,  has  the  advantage  over  the  simpler  aqueous  solution,  of 
not  so  readily  forming  insoluble  precipitates  with  albuminoids. 
— W.  T.]  How  to  proceed  so  as  to  accomplish  the  purpose  of 
the  investigation,  is  implied  in  what  has  been  written  above. 
Either  the  infected  excrement,  sputum,  or  clothing  is  exposed 
to  the  action  of  the  disinfectant  and  then  used  for  inoculation 
experiments  (e.g.,  by  inoculating  guinea-pigs  with  the  tuber- 
culous sputum  that  has  been  treated  with  carbolic  acid  or 


126  Bacteriological    TecJmology. 

•corrosive  sublimate,  or  causing-  healthy  people  to  wear  pieces 
of  clothing  disinfected  by  heat  or  chemicals) ;  or  the  micro- 
organisms mentioned  above  are  used  as  reagents,  carefully 
mingled  with  excrement  or  sputum,  or  placed  in  the  pockets 
or  under  the  lining  of  clothing  in  which  they  are  disinfected, 
and  their  power  of  development  subsequently  tested  in  the 
usual  way. 

Finally,  a  large  number  of  questions  must  be  answered 
which  are  of  a  clinical  or  technical  nature,  etc.,  and  hence  lie 
without  the  scope  of  this  work  (e.g.,  those  concerning  the  dan- 
gerousness  of  the  substance,  its  possible  injuriousness  to  the 
objects  to  be  disinfected,  offensive  odor,  expensiveness,  etc.); 
and  an  opinion  as  to  its  practical  utility  can  only  be  reached 
after  these  are  considered. 

The  rules  given  here  must  be  kept  in  mind  in  all  disinfec- 
tion experiments,  whether  referring  to  the  germicidal  power 
of  heat,  sunlight  (Duclaux),  sound  waves,  electricity,  fluids,  or 
gases.  But  the  methods  must  obviously  be  modified  in  their 
details  to  suit  each  case.  As  examples,  a  detailed  account  is 
given  here  of  the  best  manner  of  investigating  the  disinfecting 
value  of  a  fluid,  and  of  testing  a  disinfecting  oven. 

1.  TESTING  A  FLUID  DISINFECTANT. 

A.  By  the  Use  of  Fluid  Cultures.— 'Equal  quantities  of 
rather  dilute  and  more  concentrated  solutions  of  the  disinfect- 
ant are  poured  into  a  number  of  sterilized  vessels.  With  the 
same  Pasteur  pipette,  an  equal  number  of  drops  of  a  well- 
shaken  pure  culture  is  added  to  each  of  these  tubes,  and  care- 
fully mingled  with  its  contents  by  shaking.  The  time  is  noted, 
and  the  same  number  of  drops  of  the  culture  are  then  added  to 
a  control  test-tube  containing  sterile  bouillon  or  0.7  per  cent 
solution  of  table  salt. 

A  large  number  of  test-tubes,  with  5  to  10  cc.  of  nutrient 
jelly  in  each  are  kept  at  25°  to  30°  C.,  so  that  their  contents 
remain  fluid.  From  each  of  the  antiseptic  solutions  that  have 
received  bacteria,  as  well  as  from  the  control  tube,  a  drop  is 
transferred  to  one  of  the  tubes  of  jelly,  the  same  platinum 
loop  being  used  for  all,  to  insure  the  transfer  of  about  the 
same  quantity  in  each  case.  This  is  repeated  at  longer  or 
shorter  intervals  for  the  various  degrees  of  concentration,  and 


Bacteriological    Technology.  127 

the  time  for  which  the  disinfectant  was  allowed  to  act  in  each 
case,  as  well  as  its  concentration,  is  marked  upon  the  tube. 

The  appearance  or  absence  of  bacteria  in  these  isolation- 
cultures,  as  well  as  their  abundance  if  present,  give  the  data 
for  a  determination  of  the  time  required  by  the  disinfectant  in 
a  certain  degree  of  concentration,  to  destroy  a  certain  kind  of 
bacteria.  It  is  a  weak  point  in  this  mode  of  investigation, 
that  a  small  quantity  of  antiseptic  is  always  transferred  to 
the  nutrient  jelly,  together  with  the  bacteria;  but  this  can  be 
assumed  to  be  inactive  in  the  degree  of  dilution  caused  by 
mixing  it  with  the  jelly,  and  this  source  of  error  can  always 
l>e  subjected  to  the  test  of  control  experiments. 

B.  By  Using  Cultures  Dried  on  Silk,  etc. — The  investiga- 
tions are  carried  on  along  the  same  lines  as  the  last.  Sterile 
vessels  are  filled  with  solutions  of  an  antiseptic  of  various  de- 
grees of  concentration.  Several  pieces  of  silk,  charged  with 
bacteria,  are  dropped  in  each.  By  means  of  a  platinum  wire, 
pieces  are  fished  oat  at  definite  intervals,  and,  after  removal 
of  the  disinfectant,  sown  in  a  gelatinized  medium.  The  re- 
moval of  the  disinfectant  is  effected  by  carefully  pressing  the 
threads  out  in  sterile  filter-paper,  washing  them  in  water,  and 
again  pressing  them  between  folds  of  filter-paper.  The  paper 
for  this  use  is  cut  in  quadrangular  pieces  of  about  5  sq.  cm., 
folded  like  sheets  of  writing  paper.  These  are  wrapped  in 
paper  in  small  parcels,  and  sterilized  at  150°  C.  before  use. 
The  control  silk  is  placed  in  sterile  bouillon  or  0.7  per  cent  salt 
;  solution,  and  then  washed  in  the  same  manner  as  the  pieces 
that  have  been  exposed  to  the  antiseptic  solutions.  When  the 
disinfectant  has  been  thoroughly  removed  by  washing  and 
the  capillary  action  of  the  filter-paper,  the  pieces  of  silk  are 
sown  in  melted  jelly  or  on  solid  jelly.  In  the  first  case,  test- 
tubes  are  used,  which  are  rolled  or  laid  horizontally  while 
cooling,  to  facilitate  the  counting  of  the  germs.  In  the  second 
case,  watch-glasses  or  small  uncovered  trays  (Fig.  13)  are 
used.  Six  to  ten  of  these  are  placed  in  a  larger  pair  of  trays 
(Fig.  12),  sterilized  in  these  at  150°  C.,  and  only  filled  immedi- 
ately before  use.  This  arrangement  facilitates  the  microscopic 
control  of  the  cultures,  but  it  makes  later  contamination  pos- 
sible, and  requires  a  more  careful  washing  than  the  isolation 
.cultures  in  test-tubes. 

If  the  threads  are  not  sufficiently  washed  out,  the  disinfect- 


128 


Bacteriological    Technology. 


ant  remaining  in  them  may  diffuse  through  the  surrounding* 
gelatin  in  sufficient  quantity  to  prevent  all  growth  about  the 
threads,  even  when  these  contain  living  bacteria — a  fact  which 
can  cause  (and  has  caused)  mistakes.  The  thoroughness  of 
the  washing  may  be  tested  in  the  following  manner :  Some  of 
the  agar-gelatin  to  be  used  is  inoculated  with  the  same  sort  of 
bacteria  as  those  on  the  threads,  just  before  being  poured  in 
the  watch-glasses  or  trays,  where  it  is  then  allowed  to  solidify, 
and  the  silk  is  laid  on  its  surface  as  usual.  Some  days  later, 
the  colonies  appear  in  it,  reaching  quite  to  the  thread  in  case 
the  washing  has  been  sufficient,  while  a  sterile  zone  surrounds 
the  thread  if  it  has  not  been  thorough  enough. 
Attention  should  be  given  to  this  fact  in  test- 
ing the  freedom  of  antiseptic  dressings  from 
bacteria  by  sowing  them  in  nutrient  gelatin; 
control  cultures  being  made  by  means  of  a 
series  of  thrust-cultures  of  various  bacteria  in 
the  immediate  vicinity  of  the  pieces  of  mate- 
rial in  question. 

2.  TESTING  A  DISINFECTING  OVEN. 

For  this  purpose  are  needed : 

a.  A  variety  of  the  objects  the  oven  is  made 
for  (usually  bed-clothes  and  wearing-apparel) 
are  needed. 

b.  Thermometers.     Usually  it  is  sufficient 
to  have  a  few  maximum  thermometers,  which, 
to  avoid  injury  during  the  investigations,  are 

encased  in  metal  or  wood,  in  the  latter  case  openings  being* 
provided  to  prevent  them  from  checking  the  penetration  of 
the  heat.  But  if  it  is  wished  to  learn  easily  and  quickly  when 
a  certain  temperature  (e.g.,  100°  C.)  is  reached  at  a  given  point 
within  the  oven,  an  electric  contact-thermometer  Fig.  69)  is 
employed.  This  consists  of  a  thermometer  in  the  walls  of 
which  two  platinum  threads  are  fused,  one  (pi)  entering  the 
mercury  in  the  bulb,  the  other  (p)  touching  the  mercury  only 
when  the  boiling-point  of  water  (or  other  desired  temperature) 
is  reached.  ~By  means  of  screws  (s  and  Si),  the  thermometer  is 
connected  with  wires  that  may  be  passed  through  the  door 
of  the  oven  and  inserted  in  an  electric  circuit  including  a  bell 
which  sounds  when  the  circuit  is  closed. 


FIG.  69.— Electric 
Thermometer. 


Bacteriological    Technology.  1 29 

c.  Bacteria. — The  principal  of  these  are  resistant  spores, 
like  those  of  Bacillus  anthracis,  B.  subtilis,  Tyrothrix  scaber 
(Duclaux),  and  certain  earth-bacilli.  The  material  is  prepared 
as  indicated  on  pages  123-125.  For  packages,  filter-paper  is 
used,  folded  like  the  papers  used  by  druggists  for  "  powders," 
and  sterilized  at  150°  C.  The  papers  are  marked  and  num- 
bered with  a  lead -pencil,  and  those  containing  the  different 
sorts  of  spores  are  collected  in  small  parcels  which  can  event- 
ually be  tied  to  a  thermometer. 

When  the  material  for  the  test  is  ready,  the  oven  is  packed 
as  it  is  intended  to  be  in  regular  use.  The  thermometers  and 
test-bacteria  are  distributed  through  the  oven  during  this  pro- 
cess, not  merely  between  and  upon  the  clothing,  but  within  it, 
in  places  where  contagia  might  easily  exist,  but  where  the 
heat  penetrates  only  with  difficulty.  For  instance,  mattresses 
and  pillows  are  ripped  open  enough  to  allow  of  the  insertion 
of  thermometer  and  bacteria  into  the  straw,  hair,  moss  or 
feathers,  and  again  closed  by  sewing  or  the  use  of  safety  pins; 
one  or  more  blankets  are  rolled  tightly  about  thermometer 
and  bacteria,  etc.  If  the  bacteria  are  destroyed  under  these 
circumstances,  it  is  evident  a  fortiori  that  as  g-ood  results 
are  attainable  with  the  looser  packing-  which  may  be  employed 
in  the  daily  use  of  the  oven.  Care  must  be  taken  that  the 
thermometers  are  not  all  collected  about  a  single  place,  but 
are  distributed  through  the  oven. 

After  the  oven  is  packed,  it  is  best  to  make  a  diagram  (Fig-. 
70)  of  its  contents,  that  the  temperature  reached  •  and  its  in- 
fluence on  the  bacteria  may  be  subsequently  noted  at  various 
points,  giving  an  excellent  synopsis  of  the  results  of  the  test. 

At  the  end  of  the  experiment,  as  accurate  notes  as  possible 
are  made  of  the  time  for  which  it  was  continued,  the  amount 
of  fuel  consumed,  etc.;  the  articles  are  unpacked,  and  their 
appearance,  degree  of  dryness  and  brittleness,  etc.,  observed, 
and  the  thermometer  readings  made. 

The  packets  of  bacteria  are  wrapped  in  paper,  in  which 
they  can  be  carried  to  the  laboratory  without  danger  of  con- 
tamination. Cultures  are  made  from  them,  as  outlined  on  p. 
127,  either  in  test-tubes  or  trays.  But  it  is  to  be  observed 
that  the  latter  sort  of  cultures  is  not  suited  to  g-arden -earth, 
because,  even  when  the  dirt  is  poured  from  the  papers  very 
slowly,  particles  easily  fall  into  adjacent  trays.  It  must, 


130 


Bacteriological    TccJmology. 


therefore,  be  poured  on  the  surface  of  gelatin  which  has  been 
allowed  to  harden  obliquely  in  test-tubes.  In  larger  series  of 
comparative  tests,  it  is  well  to  place  the  cultures  in  a  brood- 
oven  at  20°  C.,  but  isolated  experiments  are  as  well  made  at 
the  varying-  temperature  of  the  room.  Most  cultures  require 
to  be  kept  only  a  week,  but  those  from  garden-earth  must  be 
watched  for  a  couple  of  weeks,  as  they  occasionally  contain 
resistant  forms  which  develop  very  late.  It  is  necessary  to 
note  not  only  whether  or  not  growth  occurs,  but  also  whether 
there  is  any  retardation  of  the  development  of  the  bacteria, 
and,  in  isolation  cultures,  their  number. 

The  final  results  of  the  experiment  are  most  conveniently 


FIG.  70.— Diagram  of  a  Packed  Disinfection  Oven,  with  Indication  of  the  Results  of  a  Test. 

registered  by  noting  the  thermometric  and  bacteriological  ob- 
servations upon  the  diagram  representing  the  contents  of  the 
oven.  In  Figure  70,  +  indicates  growth;  0  no  development; 
and  the  species  of  bacteria  are  indicated  by  the  relative  posi- 
tion of  the  marks,  which  are  always  arranged  in  the  same 
sequence — earth,  hay,  anthrax-bacilli.  E.g.,  within  the  upper 
mattress  of  the  Figure,  the  temperature  reached  was  102°  C., 
and  all  three  species  of  bacteria  were  killed,  while  the  earth- 
bacilli  survived  the  disinfection  within  the  rolled  woollen 
blankets,  where  the  temperature  was  between  103°  and  104° 
C.,  etc. 

As  has  been  intimated  above,  one  should  be  careful  not  to 


Bacteriological    Technology.  1 3 1 

confound  the  power  of  a  disinfectant  to  check  or  prevent  the 
growth  of  the  bacteria,  with  its  power  of  killing  them.  That  a 
substance,  added  in  a  certain  quantity  to  a  readily  putrefying 
fluid,  prevents  decomposition,  by  no  means  requires  that 
it  should  have  killed  the  bacteria  in  the  fluid.  The  development 
and  dissemination  of  the  germs  may,  perhaps,  have  been  pre- 
vented; and  it  may  be  that  when  brought  into  a  new  and  fa- 
vorable soil  they  would  prove  very  well  able  to  develop.  It  is 
not  superfluous  to  mention  this  perfectly  obvious  fact,  because 
it  is  often  overlooked,  giving  origin  again  and  again  to  errors. 

If  it  is  wished  to  learn  the  degree  of  concentration  in  which 
a  given  material  begins  to  check  the  growth  of  different 
micra  organisms,  and  that  in  which  it  renders  growth  entirely 
impossible,  the  following  method  is  employed,  bacteria  and 
moulds  being  used,  selected  as  indicated  on  p.  122. 

A  suitable  number  of  test-tubes  are  filled  with  nutrient 
material  (generally  gelatin)  adapted  to  the  organisms  selected. 
Some  of  these  glasses  are  left  untouched,  serving  for  control, 
while  larger  or  smaller  quantities  of  the  substance  to  be  tested 
are  added  to  the  rest,  so  that  all  degrees  of  concentration  are 
obtained.  In  preliminary  experiments,  it  is  best  to  pour  nearly 
equal  quantities  of  gelatin  into  all  of  the  tubes,  adding  to  them 
different  quantities  of  a  strong  solution  of  the  substance,  of 
known  concentration,  by  means  of  glass  pipettes  graduated  to 
0.05  cc.  From  these  data,  the  percentage  of  the  substance  in 
each  culture  glass  can  readily  be  calculated. 

The  species  of  bacteria  on  which  it  is  desired  to  test  the 
disinfectant  is  sown  in  the  vessels  prepared  in  this  way,  the 
material  used  for  inoculating  all  being  taken  from  a  single 
culture  and  by  means  of  the  same  needle  or  pipette.  The 
quantity  used  for  inoculation,  and  the  manner  in  which  the 
needle  is  used  should  also  be,  as  far  as  possible,  exactly  the 
same  for  the  different  glasses,  which  are  then  observed  daily, 
the  least  degree  of  concentration  which  permits  no  growth 
along  the  thrust  and  the  least  which  perceptibly  checks  the 
growth  of  the  bacteria,  being  noted.  The  latter  point  is  chiefly 
indicated  by  a  retardation  of  development,  and  by  a  change 
in  the  macroscopic  habit  of  the  colonies. 

Figure  71  illustrates  this.  It  represents  three  test-tubes 
with  peptonized  meat-infusion  gelatin  to  which  0.05  per  cent  of 
carbolic  acid  was  added  in  the  first,  0.4  per  cent  in  the  second, 


I32 


Bacteriological    Technology. 


and  0.7  per  cent  in  the  third.  The  same  bacillus  was  sown  in 
all  three  glasses,  which  eight  days  later  presented  the  appear- 
ance represented.  In  the  first  tube,  the  appearance  of  the 
culture  was  the  same  as  in  the  untreated  control-tube — a  large 
close  irregular  felt  of  bacilli  floating-  on  a  quantity  of  melted 
g-elatin  at  the  very  top,  and  below,  above  the  thrust  in  the 
still  solid  gelatin,  a  number  of  dot-like  colonies,  from  some  of 
which,  especially  the  uppermost,  fine  undulating1  threads  radi- 
ate in  all  directions.  In  the 
second  tube,  during  the  same 
time,  the  felt  of  bacilli  next  the 
surface  had  not  reached  the 
glass,  liquefaction  was  limited 
to  a  small  hemispherical  de- 
pression immediately  about 
the  needle-thrust,  while  no 
trace  was  to  be  seen  of  fila- 
ments from  the  deeper  colo- 
nies, which  did  not  approach 
the  bottom  so  nearly  as  in  the 
first  glass.  The  third  tube 
showed  little  surface  growth, 
no  liquefaction,  and  only  a 
short,  firm,  inversely  conical 
colony  in  the  solid  gelatin. 
Very  similar  differences  are 
found  in  cultures  of  Koch's 
bacillus  of  mouse  septicaemia, 
grown  in  g-elatin  with  and 

.  °  .  FIG.  71.— Three  Cultures  of  B.  anthracis  after 

Without      Carbolic     add.        The  Seven  Days1  Growth  in  Peptonized  Meat  Gela- 

peculiar  cloud-like  fine  growth  tine' to  which  has  been  Added  the  Percentage 

of  Carbolic  Acid  Indicated  in  Each. 

never  appears  in  the  cultures 

containing  much  carbolic  acid,  in  which  the  bacilli  show  as 

small,  dense,  dot-like  colonies. 

This  prepares  the  observer  for  morphological  changes  in 
the  microscopic  appearance  of  the  bacteria  so  grown;  and  it 
must  also  be  remembered  that  under  such  conditions  a  perma- 
nent physiological  transformation  of  the  bacteria  has  been 
induced,  as  in  the  attenuation  of  the  virulence  of  B.  anthracis 
by  cultivating  it  in  carbolic  acid  (Toussaint,  Chamberland,  and 
Roux) — a  result  which  gives  especial  interest  to  experiments 
of  this  sort. 


CHAPTER  XIII. 

MICROSCOPIC  EXAMINATION,  AND  STAINING  OF  BACTERIA. 

ONE  side  of  the  microscopic  examination  of  bacteria  has 
already  been  considered  in  Chapter  IX.  (moist  chambers),  and 
in  sketching  the  plate-cultures  of  Koch,  where  not  only  the 
peculiarities  of  the  colonies  (whether  entire,  lobed,  fringed, 
smooth,  granular,  wrinkled,  etc.)  can  be  observed  with  weaker 
and  medium  powers,  but  which  without  further  contrivances, 
may  also  be  studied  with  higher  powers,  the  most  superficial 
colonies  being  even  accessible  to  the  most  powerful  immersion 
lenses  when  covered  with  a  cover-glass. 

If  it  is  wished  to  study  individual  cells  more  closely,  the 
colony  need  only  be  touched  with  a  sterile  platinum  needle, 
and  the  adhering  material  distributed  through  a  small  drop 
of  0.7-per-cent  solution  of  table  salt  (free  from  bacteria),  spread 
in  a  thin  layer  under  a  cover-glass.  If  the  bacteria  grow  in  a 
fluid  medium,  a  small  drop  of  the  culture  is  placed  under  the 
cover-glass  without  further  treatment  except  for  final  dilation 
with  the  standard  salt  solution.  The  addition  of  such  a  neu- 
tral fluid  may  occasionally  facilitate  the  investigation,  not 
only  by  isolating  the  germs,  but  by  modifying  the  differences 
of  refraction. 

If  such  preparations  are  to  be  examined  for  a  longer  time 
or  with  an  immersion  lens,  it  should  be  sealed  with  paraffin, 
which  is  best  applied  by  means  of  a  glass  tube  4  mm.  in  diam- 
eter tilled  with  paraffin  and  drawn  out  to  a  short  open  point, 
from  which  the  melted  paraffin  flows. 

The  movements  of  living-  bacteria  cannot  be  observed  long1 
in  such  sealed  preparations,  since  they  are  stopped  by  want  of 
oxygen.  This  may  be  put  off  for  a  time  by  having-  a  number 
of  rather  larg-e  air-bubbles  under  the  cover-glass,  or,  in  case 
the  bacteria  are  contained  in  water,  by  introducing  one  of  the 
filamentous  green  algae  (Engelmann).  Impelled  by  their  need 


1 34  Bacteriological    Technology. 

of  oxygen,  the  bacteria  collect  about  the  bubbles  or  algae  (the 
latter  of  which  continue  to  set  free  oxygen,  as  a  result  of  as- 
similation), and  keep  up  their  motions  until  the  supply  is  ex- 
hausted or  the  activity  of  the  algge  ceases.  The  study  of  the 
manner  in  which  the  bacteria  move  can  be  facilitated  by 
slightly  coloring  them,  as  indicated  below.  The  movements 
of  the  very  active  species  can  be  checked  by  compressing  them 
strongly  by  pressure  on  the  cover-glass.  All  motion  in  a  lim- 
ited part  of  the  preparation  may  be  stopped,  by  the  same 
means,  individuals  here  and  there  afterward  recommencing 
their  movement,  but  at  first  very  slowly. 

The  best  way  of  studying  the  movements  of  bacteria  is, 
naturally,  by  the  use  of  a  moist  chamber,  Ranvier's  moist 
chamber,  and  the  method  indicated  on  p.  98  is  especially 
advisable,  as  the  difficulties  of  using  the  hanging  drop  are 
thus  avoided. 

The  use  of  staining  fluids,  especially  certain  aniline  colors, 
is  an  indispensible  adjunct  in  the  microscopic  study  of  bac- 
teria. The  employment  of  the  methods  of  staining  now  used, 
not  only  greatly  facilitates  the  demonstration  and  observation 
of  bacteria  in  fluids,  but  the  detection  of  structural  peculiari- 
ties which  escape  observation  in  the  unstained  cells,  e.g.,  the 
flagella  of  certain  bacteria,  the  peculiar  ends  of  the  cells  of 
B.  anthracis,  etc. ;  and  it  also  discloses  chemical  differences 
which  are  sometimes  of  extraordinary  importance  for  clinical 
diagnosis,  e.g.,  in  demonstrating  the  tubercle  bacillus,  stain- 
ing has  also  rendered  it  possible  to  obtain  handsome  perma- 
nent preparations  of  free  bacteria,  and,  above  all,  to  demon- 
strate bacteria  within  the  tissues  and  to  study  their  distribution 
in  the  organs  and  their  relations  to  the  cells.  The  develop- 
ment of  the  technology  of  staining  to  the  high  point  it  has 
now  reached  is  due  chiefly  to  three  men,  Weigert,  Koch,  and 
Ehrlich. 

The  dyes  used  in  bacteriological  investigation  nearly  all 
belong  to  the  large  class  of  aniline  colors,  and  especially  to 
the  group  called  basic  by  Ehrlich,  who  first  called  attention 
to  the  fact  that  the  aniline  colors  are  divisible  into  two  main 
groups :  the  acid  colors,  in  which  the  staining  principle  is  an 
acid  (e.g.,  ammonium  picrate);  and  the  basic,  consisting  of  a 
staining  base  in  combination  with  an  acid  that  does  not 
stain  (e.g.,  acetate  of  rosaniline).  Methylene  blue,  fuchsin,. 


Bacteriological    Technology.  1 3  5 

• 

gentian-violet,  vesuvin,  etc.,  belong-  to  this  group  of  basic 
dyes  (used  by  liistologists  as  nuclear  stains),  which  have 
proved  especially. adapted  to  the  staining-  of  bacteria,  free  or 
in  situ,  so  that  it  has  proved  possible  by  their  use  to  stain  all 
known  pathogenic  bacteria  in  sections,  strongly,  permanently, 
sparingly,  and  distinct  from  the  invaded  tissue.  Ehrlich  like- 
wise calls  those  dyes  neutral  which  are  formed  by  the  union 
of  a  base  and  acid  both  of  which  are  capable  of  staining  (e.g., 
picrate  of  rosaniline). 

Naturally,  it  is  not  always  possible  to  at  once  secure  all  of 
these  desirable  results  equally  well,  and  especially  ,soft  stain- 
ing of  the  histological  elements  is  often  advantageously  made 
to  give  place  to  the  intense  coloring-  of  all  bacteria  present. 
But  Baumgarten  has,  for  instance,  succeeded  by  a  rather  com- 
plicated method  in  showing  upon  the  same  slide  the  tubercle- 
bacilli  and  the  caryokineses  resulting-  from  their  invasion. 
Baumgarten's  method  is  the  following:  The  diseased  rabbit  is 
killed,  and  small  pieces  of  the  tuberculous  organs  are  cut  out 
as  quickly  as  possible  and  at  once  cast  into  0.2-per-cent  chromic 
acid,  where  they  are  hardened  for  forty-eight  hours,  then 
washed  out  thoroughly  in  running  water  for  as  much  as 
twenty-four  hours,  and  rehardened  for  twenty -four  hours  in 
strictly  absolute  alcohol.  The  thinnest  possible  sections  are 
laid  in  freshly  prepared  aniline-methyl  violet  (No.  8)  for  a  long- 
time (as  much  as  forty-eight  hours),  washed  not  to  exceed 
thirty  seconds  in  1  part  of  nitric  acid  to  5  of  water,  the  de- 
colorization  being  then  completed  in  60-per-cent  alcohol  (No. 
17),  after  which  they  go  into  a  mixture  of  equal  parts  of  a 
concentrated  alcoholic  solution  of  fuchsin  (No.  1)  and  distilled 
water  for  half  an  hour  to  an  hour,  then  for  five  to  ten  seconds 
in  an  aqueous  solution  of  methylene  blue  (1  : 1,000),  and  finally 
for  five  to  ten  minutes  in  absolute  alcohol,  changed  once  or 
twice.  The  sections  are  finally  mounted  in  balsam  thinned 
with  bergamot  oil,  but  without  chloroform. 

Instead  of  the  more  complicated  method  of  staining  the 
nuclei  with  fuchsin  and  methylene  blue,  according-  to  Baum- 
garten a  concentrated  solution  of  vesuvin  in  1-per-cent  acetic 
acid  may  be  used. 

For  securing-  intense  and  permanent  staining,  the  same 
means  are  employed  as  in  dyeing1  on  a  larger  scale;  i.e.,  by 
the  prolonged  action  of  the  staining-  fluid;  warming-  the  lat- 


1 36  Bacteriological    Technology. 

ter,  either  for  a  long-  time  in  the  thermostat  at  40°  to  50°  C., 
or  more  intensely  but  for  a  shorter  time  over  the  flame;  or  by 
the  use  of  mordants,  e.g., — potash,  carbolic  acid,  aniline  oil,  or 
tannin — which  have  a  certain  tendency  to  unite  with  both  the 
dye  and  the  object  to  be  stained,  so  that  they  serve  as  a  sort 
of  connecting-  link. 

The  possibility  of  staining'  bacteria  in  contrast  with  the 
surrounding-  tissue  is  partly  due  to  the  different  "elective" 
power  of  various  dyes  (Ehrlich),  i.e.,  their  different  power  of 
staining- certain  tissue  elements;  and  partly  to  the  different 
strength  with  which  they  stain  various  parts  of  the  prepara- 
tion, i.e.,  the  different  stability  of  the  union  they  form  with 
them.  The  following-  experiment  of  Ehrlich  and  Schwarze 
illustrates  in  a  striking-  way  the  different  elective  power  of 
three  acid  aniline  colors,  aurantia,  indulin,  and  eosin.  When 
a  cover-glass  preparation  of  blood  is  made  by  distributing  it 
in  a  thin  layer,  drying-  it,  and  heating  to  120°  C.,  and  the  pig- 
ments are  tested  on  it,  it  is  seen  that  each  of  the  latter  pos- 
sesses the  power  of  coloring-  the  red  corpuscles  as  well  as  the 
nuclei  of  the  white  corpuscles  and  the  peculiar  granules,  found 
in  some  of  the  white  cells,  which  Ehrlich  calls  a-granules. 
But  if  the  blood-preparation  is  treated  with  a  solution  of  the 
three  dyes  in  glycerin  (made  by  adding-  an  excess  of  eosin  and 
indulin  to  a  mixture  of  1  part  of  saturated  solution  of  aurantia 
in  glycerin,  and  2  parts  of  pure  glycerin),  the  hsemoglobin  is 
colored  yellow  by  the  aurantia  ;  the  nuclei,  gray  or  black  by 
the  indulin,  and  the  a-granules,  red  by  the  eosin.  The  fact 
that  certain  parts  of  the  preparation  hold  the  coloring-  matter 
more  tenaciously  than  others,  is  used  in  demonstrating  bac- 
teria in  sections,  the  g-eneral  plan  being  to  overstain  the  entire 
preparation,  subsequently  securing-  the  differentiation  by  de- 
colorizing (and  finally  staining  in  some  contrasting  color)  cer- 
tain parts,  leaving  the  others  colored  as  at  first.  The  degree 
of  decolorization  can  be  regulated  by  the  substance  chosen  to 
effect  it.  If,  for  example,  we  have  a  section  of  an  organ  con- 
taining bacteria,  which  has  lain  in  a  strong  solution  of  methyl 
violet,  and  wash  it  in  water,  microscopic  examination  will 
show  the  bacteria,  as  well  as  the  protoplasm  and  nuclei  of  the 
cells,  colored  deep  violet  (diffuse  staining).  If  it  is  washed  in 
dilute  acetic  acid  (p.  141,  No.  11),  the  protoplasm  becomes  de- 
colorized, while  the  bacteria  and  nuclei  retain  the  color 


Bacteriological    Technology.  137 

(nuclear  staining-).  If  it  is  washed  in  potassium  carbonate 
(No.  15),  the  coloring-  matter  is  also  driven  from  the  nuclei,  the 
bacteria  alone  remaining  stained  (isolated  staining-  of  the  bac- 
teria). By  still  more  powerful  ag-ents,  such  as  25-per-cenfc 
nitric  acid  or  hydrochloric  acid,  most  species  of  bacteria  can 
likewise  be  decolorized,  so  that  only  a  very  few  sorts  remain 
stained  (tubercle  stain) ;  but  repeated  treatment  with  strong1 
solutions  of  mineral  acids,  if  continued  sufficiently  long-,  also 
removes  the  stain  from  these  species,  and  it  must  be  remem- 
bered throughout  that  the  time  for  which  the  preparations 
are  exposed  to  the  action  of  the  decolorizing  agent  is  of  de- 
cisive importance  for  the  result,  since  the  power  of  the  differ- 
ent elements  to  resist  decolorization  usually  differs  only  in 
degree,  not  in  kind.  In  each  case,  therefore,  it  is  necessary  to 
avoid  decolorizing  too  little  or  too  much,  and  as  it  is  not  easy 
to  give  very  exact  instructions  as  to  the  time  needed,  this  is 
one  of  the  points  on  which  long-  experience  plays  an  import- 
ant part. 

To  further  differentiate  the  bacteria  from  the  tissue  ele- 
ments, and  to  bring  the  form  and  disposition  of  the  latter  out 
more  clearly,  multiple  staining  (usually  double  staining-)  can 
be  employed,  either  by  using  a  new  staining-  fluid  on  the  partly 
decolorized  preparations  (successive  staining-,  e.g.,  No.  17), 
or  by  treating  them  with  a  fluid  which  at  once  decolorizes  and 
restains  them  in  part  (replacement  staining).  Treating-  the 
same  preparation  with  two  colors  is  also  sometimes  resorted  to 
for  another  purpose,  namely  to  render  the  staining  more  re- 
sistant toward  decolorizing  substances,  either  by  forming  a 
new  compound  color,  or  otherwise,  e.g.,  the  gentian-violets 
iodine  method  (Gram)  and  the  fuchsin-methylene-blue  method 
described  on  p.  147. 

Occasionally  double  staining  may  be  secured  by  the  em- 
ployment of  a  single  dye,  e.g.,  when  methylene  blue  is  used  to 
stain  a  section  which  includes  bacteria  as  well  as  the  pecu- 
liarly granulated  connective-tissue  cells  known  as  "Mastzel- 
len,"  the  bacteria  are  colored  blue,  and  the  granules  violet. 
These  granules  (the  ^-granules  of  Ehrlich)  also  deserve  notice 
for  another  reason.  Since,  like  bacteria  and  nuclei,  they  are 
stained  by  basic  aniline  colors,  and  have  about  the  size  of 
micrococci,  they  may  be  mistaken  for  the  latter,  as  has  often 
been  the  case  with  inexperienced  observers.  As  an  especially 


138  Bacteriological    Technology. 

favorable  object  for  the  comparative  study  of  micrococcus 
cells  and  these  /'-granules,  I  can  recommend  the  mesentery  of 
a  rather  lean  mouse,  dead  of  suppurative  peritonitis  as  a  result 
of  inoculation  with  pyogenic  staphylococci.  The  mesentery  is 
spread  out  on  a  cover-glass  which  is  slipped  beneath  it  while 
it  is  still  attached  to  the  intestine,  by  the  weight  of  which  it 
is  stretched  over  the  glass.  As  soon  as  it  has  dried  fast,  the 
part  projecting  around  the  edge  of  the  glass  is  cut  away,  and 
it  is  treated  as  if  it  were  an  ordinary  cover-glass  preparation 
(by  drying,  passing  through  the  flame,  etc.),  and  stained  by 
methylene  blue.  Among  the  blue  cocci  will  be  found  small 
clusters  of  granules  of  unequal  size,  grouped  irregularly 
around  an  unstained  nucleus — for  the  nuclei  of  these  cells, 
unlike  those  of  other  connective-tissue  cells,  are  not  colored 
by  this  mode  of  staining  with  basic  aniline  colors.  In  double- 
staining,  colors  are  naturally  chosen  which  contrast  sharply 
and  prettily  with  one  another — e.g.,  fuchsin  and  methyl  green; 
fuchsin  and  methyl  blue;  methyl  violet  and  vesuvin  [or 
methyl  violet  and  eosin]. 

An  extremely  large  number  of  dyes  have  gradually  come 
into  use  in  various  ways  for  staining  bacteria ;  but  only  a  lim- 
ited number  of  stains  and  methods  will  be  considered  here, 
and  it  is  best,  for  beginners  especially,  to  be  content  with  one 
or  two  of  the  most  universal  and  important  methods,  practising- 
them  carefully  until  they  are  mastered,  before  going  further. 
It  is  not  possible  to  limit  one's  self  exclusively  to  a  single  dye, 
for  the  reason  that  the  behavior  of  different  bacteria  toward 
the  various  staining  fluids,  and  when  different  methods  are 
used,  is  of  diagnostic  importance,  while  there  is  no  one  dye 
which  stains  all  bacteria  equally  well.  E.g.,  the  slight  power  of 
methylene  blue  to  stain  the  bacillus  of  leprosy  can  be  used  to 
distinguish  the  latter  from  that  of  tubercle,  which  otherwise 
resembles  it  closely  (c/.  p.  155).  In  other  cases,  the  behavior 
when  the  Gram  method  is  employed  can  be  utilized  for  diag- 
nosis; e.  g.,  with  gonococci,  which  (like  the  typhoid  bacillus, 
the  cholera  spirillum,  and  the  microbes  of  chicken  cholera  and 
the  septicaemia  of  mice)  are  decolorized  when  the  Gram  method 
is  used,  while  other  pyogenic  micrococci,  which  closely  resem- 
ble them,  retain  the  stain.  The  most  nearly  universal  pig- 
ment is  methylene  blue,  a  substance  introduced  by  Ehrlich, 
which  can  well  be  used  as  the  principal  stain.  By  employing- 


Bacteriological    Technology.  1 39 

it  in  the  manner  indicated  below  (No.  XIII.),  Kiihne  succeeded 
in  staining  in  situ  all  of  the  bacteria  that  he  investigated.  In 
leprosy  and  mouse  septicaemia,  alone,  he  failed  to  obtain  com- 
pletely satisfactory  results/and  for  the  bacilli  of  these  diseases 
he  employed  fuchsin.  Consequently,  it  is  best  to  carefully 
practise  this  "  universal  method  with  methylene  blue ; "  and  if 
to  this  are  joined  one  of  the  tubercle  methods  (e.g.,  No. 
XVIII.),  and  the  Gram  method,  one  is* well  equipped,  so  far  as 
sections  are  concerned.  It  is  also  best  to  confine  one's  self  to 
a  few  methods  for  cover-glass  preparations. 

The  materials  employed,  in  the  methods  described  below, 
are  the  following :  Distilled  water,  absolute  alcohol,  glycerin, 
acetic  acid,  hydrochloric  acid,  nitric  acid,  sulphuric  acid, 
chromic  acid,  carbolic  acid,  aniline  oil,  potash,  potassium  car- 
bonate, potassium  acetate,  potassium  iodide,  lithium  carbon- 
ate, iodine,  clove  oil,  bergamot  oil,  cedar  oil,  turpentine,  xylol, 
Canada  balsam,  shellac,  paraffin,  fuchsin,  methylene  blue, 
methyl  violet,  gentian  violet,  methyl  green,  vesuvin,  picro-car- 
mine,  and  extract  of  logwood.  Different  aniline  colors  bearing 
the  same  name  may  differ  to  such  an  extent  as  not  to  be 
equally  adapted  to  histological  purposes,  so  that  attention 
should  be  given  to  the  source  and  trade  mark  of  good  sorts. 

All  of  the  aniline  colors  named  should  be  kept  in  stock  in 
the  dry  form.  Fuchsin,  methylene  blue,  methyl  violet,  and 
gentian  violet  may  be  further  kept  in  a  saturated  alcohol 
solution  (No.  1),  i.e.,  25  gm.  of  the  dye  to  100  gm.  of  absolute 
alcohol,  which  always  leaves  an  abundant  excess  of  undis- 
solved  pigment  in  the  bottom  of  the  flask.  Vesuvin  (or  Bis- 
marck brown)  is  best  kept  only  in  powder,  but  if  a  solution  is 
to  be  kept,  this  is  best  made  (No.  2)  by  saturating  equal  parts 
of  water  and  glycerin. 

For  use,  aqueous  solutions  of  the  powders  (No.  3)  may  be 
directly  prepared,  but  these  do  not  keep  long,  and  are  there- 
fore always  freshly  prepared  from  water  free  from  bacteria, 
and  are  filtered  before  use.  It  is  very  convenient  to  keep 
a  little  of  the  dry  pigment  constantly  used,  in  this  way,  on  a 
little  filter  in  a  glass  funnel.  When  it  is  needed  a  small 
quantity  of  water  is  poured  over  it,  and  the  filtrate  is  collected, 
the  powder  soon  drying  again.  Dust  is  kept  from  it  by  cover- 
ing the  funnel  with  a  layer  of  filter-paper. 

When  it  is  not  exceptionally  necessary  to  avoid  every  trace 


140  Bacteriological    Technology. 

of  alcohol  in  the  staining-  fluid  (as  is,  for  example,  the  case 
when  staining-  living-  bacteria),  the  aqueous  solution  is  re- 
placed without  disadvantag-e  by  that  (No.  4)  made  by  adding  a 
suitable  quantity  of  the  saturated  alcoholic  solution  (No.  1)  to 
water.  This  diluted  alcoholic  solution  is  more  durable  than 
the  aqueous,  but  it  usually  requires  renewal  once  or  twice  a 
month,  so  that  it  is  best  to  prepare  it  when  needed,  by  adding 
five  to  six  drops  of  No.  1  to  a  watch-glass  full  of  distilled  water. 

The  principal  mordants  used  are  0.01-per-cent  solution  of 
potash  (Koch,  Loeffler) :  5-per-cent  carbolic  acid  (Ziehl) ;  and 
aniline-water  (No.  5),  a  concentrated  aqueous  solution  of  ani- 
line oil,  prepared  by  very  thoroughly  shaking  about  1  part  of 
aniline  oil  and  20  parts  of  distilled  water  in  a  test-tube,  allow- 
ing- it  to  stand  five  minutes,  and  filtering-  through  a  filter 
moistened  with  distilled  water.  (It  must  be  perfectly  free 
from  turbidity,  or  it  should  be  again  shaken,  and  refiltered.) 
The  staining  fluids  with  mordants  for  which  we  shall  find 
application,  are : 

(No.  6.)  Kuehne's  carbolic  blue.  1.5  parts  of  methylene 
blue,  and  10  parts  of  absolute  alcohol  are  triturated  lightly  in 
a  watch-glass  with  100  parts  of  5-per-cent  carbolic  acid  which 
is  added  little  by  little.  When  all  is  dissolved,  it  is  bottled. 
To  facilitate  rapid  preparation,  several  test-tubes  with  feet 
may  be  graduated  to  20,  22,  and  24.2  cc.  (cf.  Fig.  72). 

(No.  7.)  Ziehl's  carbolic  fuchsin.  1  part  of  fuchsin,  10  of 
alcohol,  and  100  of  5-per-cent  carbolic  acid. 

(No.  8.)  Aniline  methyl  violet  (Ehrlich-Weigert).  11  cc.  of  the 
saturated  alcoholic  solution  of  methyl  violet,  10  cc.  of  absolute 
alcohol,  and  100  cc.  of  aniline-water.  Or  the  dry  powder  may 
be  added  in  excess  to  aniline  water.  [In  Koch's  laboratory  it 
is  customary  to  add  the  stock  alcoholic  solution  (No.  1)  to  a 
watch-glass  of  aniline-water  until  the  latter  is  shown  to  be 
saturated  by  the  formation  of  a  film  at  top.] 

(No.  9.)  Aniline  gentian  violet  (Ehrlich).  5  cc.  of  the  satu- 
rated alcoholic  solution  of  gentian  violet,  to  100  cc,  of  aniline- 
water. 

(No.  10.)  Loeffler's  alkaline  blue.  30  cc,  of  the  saturated 
alcoholic  solution  of  methylene  blue  to  100  cc.  of  0.01-per-cent 
caustic-potash  solution. 

A  large  part  of  the  chemicals  enumerated  above  are  used 
for  washing  preparations  for  decolorization  or  differentiation. 


Bacteriological    Technology.  141 

For  this  purpose,  water,  alcohol,  and  glycerin  (all  perfectly, 
free  from  acid)  are  used,  as  well  as  clove-oil  and  aniline-oil, 
and  acids  and  salts  in  the  following-  forms : 

(No.  11.)  Very  dilute  acetic  acid  (0.5  per  cent  to  1  per  cent). 

(No.  12.)  Very  dilute  hydrochloric  acid  (10  drops  of  the 
acid  to  500  gm.  water). 

(No.  13.)  75  parts  of  water  containing-  25  parts  of  nitric 
acid  (Ehrlich),  or  the  same  quantity  of  hydrochloric  or  sul- 
phuric acid. 

(No.  14.)  Lithium  water  (Kuehne).  6  to  8  drops  of  a  con- 
centrated aqueous  solution  of  lithium  carbonate  and  10  gm. 
water.  For  neutralizing-  an  acid  washing-  fluid. 

(No.  15.)  Potassium  carbonate  solution  (Koch).  Equal 
parts  of  a  saturated  aqueous  solution,  and  water  (Koch) ;  or  2 
parts  of  a  2-per-cent  aqueous  solution,  and  1  part  of  absolute 
alcohol  (Malassez  and  Vig-nal). 

(No.  16.)  Gram  solution.  Iodine  1  part;  potassic  iodide,  2 
parts;  distilled  water,  300  parts. 

(No.  17.)  Alcohol,  60  parts;  water,  40  parts  (Koch).  For 
colored  wash -alcohol  (Kuehne),  see  p.  151. 

(No.  18.)  Aniline-oil  blue.  Kuehne  recommends  rubbing  as 
much  methylene  blue  as  can  be  raised  on  the  point  of  a  knife, 
with  10  gm.  clarified  aniline  oil,  and  allowing-  the  excess  of  un- 
dissolved  pigment  to  settle  in  a  bottle.  A.  few  drops  are  added 
to  aniline  oil  in  a  watch-g-lass,  until  the  desired  concentration 
is  reached. 

The  manner  of  using  these  staining  and  decolorizing-  ag-ents, 
as  well  as  the  others  for  hardening-,  anhydrating-,  clearing, 
mounting-,  and  sealing1  preparations,  receives  more  detailed 
consideration  under  the  several  methods  described  below; 

In  what  follows,  it  is  assumed  that  the  common  micro- 
scopic appliances  and  methods  are  understood.  Staining  is 
usually  effected  in  small  glass  trays  (Fig-.  13),  "individual  salt 
cellars"  or  watch-glasses.  Some  of  the  trays  must  have 
ground  tops  so  that  they  can  be  tightly  sealed  with  a  glass 
plate  if  they  are  to  kept  at  an  elevated  temperature  for  some 
time.  Watch-glasses,  which  are  inconvenient  because  of  their 
lack  of  stability,  are  necessary  if  sections  are  to  be  stained  by 
heating  over  the  flame.  In  this  case,  they  are  conveniently 
placed  upon  the  stand  shown  in  Figure  72,  which  consists  sim- 
ply of  a  strip  of  sheet  tin  with  three  holes  having  a  somewhat 


142  Bacteriological    Technology. 

smaller  diameter  than  the  watch-glasses.     The  stand  is  also 
useful  for  filtering,  as  indicated  in  the  cut. 

STAINING  BACTERIA  IN  FLUIDS. 

A.  By  the  Simple  Addition  of  the  Staining  Fluid.— A  lit- 
tle drop  of  very  dilute  aqueous  solution  (No.  3)  of  fuchsin  or 
methyl  green  (Mace)  is  placed  on  a  slide,  a  small  quantity  of 
the  culture,  etc.,  is  distributed  through  it  with  a  platinum 
needle,  and  a  cover-glass  applied.  The  staining  fluid  must 
usually  be  so  dilute  that  it  appears  nearly  colorless  under  the 
microscope.  When  pure  and  extremely  dilute  aqueous  solu- 


FIG.  72.— Tin  Support  for  Funnels  and  Watch-glasses,  in  Staining  and  Filtering. 

tions  of  fuchsin  are  used,  the  bacteria  can  remain  alive  and 
continue  their  motions  after  staining  (Salomonsen). 

If  a  drop  of  the  culture  and  one  of  a  more  concentrated  stain- 
ing fluid  than  in  the  last  case  are  placed  in  proximity. on  the 
slide  and  covered  with  a  single  cover-glass,  a  series  of  bacteria 
may  be  found  in  the  same  preparation,  showing  all  gradations 
from  cells  that  are  unstained  to  others  strongly  overstained. 

A  rapid  microscopic  view  of  a  large  number  of  crowded 
colonies  in  a  plate -culture  is  obtained  by  the  following  method : 
A  well-cleansed  cover-glass  is  laid  upon  the  gelatin  over  the 
colonies  and  pressed  firmly  into  contact  with  it  everywhere, 
by  a  glass  rod  or  pair  of  forceps.  Fragments  of  the  super- 
ficial colonies  adhere  to  the  cover,  forming  an  "impression- 
preparation  "  of  the  culture,  so  that  when  it  is  removed  from 
the  gelatin  and  lowered  on  to  a  drop  of  staining  fluid  on  a 


Bacteriological    Technology.  143 

slide,  the  opportunity  is  given  to  see  small  samples  of  the  con- 
tents of  all  of  the  colonies  touched.  Any  of  these  preparations 
can  be  sealed  with  paraffin,  as  described  above. 

B.  Staining  Preparations  Dried  on  the  Cover  glass. — 
Weigert  was  the  first,  in  1876,  to  recommend  an  aniline  color 
(methyl  violet)  for  staining1  bacteria.  Shortly  afterward,  with- 
out knowing-  of  Weigert's  work,  I  introduced  fuchsin  as  par- 
ticularly good  for  staining-  bacteria,  and  indicated  especially 
its  value  as  a  means  of  diagnosis  in  the  microscopic  analysis 
of  putrefying-  blood.  By  the  introduction  of  the  basic  aniline 
colors,  the  demonstration  and  examination  of  bacteria  in  fluids 
was  greatly  facilitated;  but  a  further  and  decisive  advance  in 
methods  was  made  in  1878,  when  Koch  showed  how  bacteria 
might  be  colored  after  drying  them  in  a  very  thin  layer  upon 
the  cover-glass,  which,  likewise,  rendered  possible  the  complex 
micro-chemical  treatment  of  free  bacteria  which  now  plays  so 
important  a  part.  The  great  advantages  of  this  method  have 
caused  it  to  be  almost  exclusively  used  in  all  chemical  examin- 
ations of  blood,  pus,  urine,  and  sputum ;  consequently  the  mak- 
ing and  examining  stained  cover-glass  preparations  will  be 
sketched  in  detail  in  the  following  pa.ges.  Obviously,  the  use 
of  such  preparations  does  not  render  superfluous  the  simple 
staining-  already  described,  since  the  latter  completely  pre- 
serves the  form  and  turgescence  of  the  cells,  while  a  shrinking 
always  results  from  drying  them. 

New  cover-glasses  are  carefully  washed  in  warm  water, 
dried,  and  treated  with  absolute  alcohol  for  the  removal  of  all 
grease.  Those  that  have  been  used  are  first  laid  in  strong- 
mineral  acid  (hydrochloric  or  sulphuric),  then  washed  clear  in 
water  and  rinsed  until  every  trace  of  acid  is  removed,  when 
they  are  treated  as  if  new. 

The  fluid  is  best  spread  in  a  thin  layer,  by  placing-  a  small 
drop  on  a  cover-glass,  laying  another  cover  upon  it,  and  sepa- 
rating them  by  drawing-  one  over  the  other,  in  which  way  two 
preparations  are  obtained.  It  is  also  possible  to  scrape  the 
fluid  in  as  thin  a  layer  as  possible  by  the  edge  of  a  second 
cover-glass.  The  covers  are  then  laid  to  dry  under  a  bell- 
glass,  with  the  smeared  side  up.  If  the  drying  is  to  be  accel- 
erated by  warming,  this  must  be  effected  at  a  very  low  tem- 
perature. The  air-drying  must  be  complete  before  the  next 
step  is  taken. 


144  Bacteriological    Technology. 

When  such  a  dried  preparation  of  blood,  pus,  sputum,  or 
other  fluid  which  contains  albumen,  is  at  once  stained,  two 
defects  are  sometimes  observed ;  sometimes  the  dried  film  sep- 
arates in  part  from  the  glass;  sometimes  the  presence  of  sol- 
uble albuminoids  causes  disturbing  precipitates  in  the  prepa- 
ration. These  are  avoided  when  the  albuminoids  are  rendered 
insoluble  by  suitable  hardening'.  The  second  defect  can  also  be 
avoided  by  using1  aniline  brown  (Bismarck  brown  or  vesuvin) 
in  glycerin  (No.  2),  and  washing1  in  pure  glycerin  (Koch). 
Absolute  alcohol  (Koch)  may  be  used  for  this  purpose,  the 
cover-glass  being-  placed  in  it  for  some  time,  but  the  requisite 
time  varies  much  for  different  preparations.  Partly  for  this 
reason,  and  partly  because  of  its  slowness,  this  method  is  far 
inferior  to  that  of  Ehrlich,  by  heating-  up  to  120°  to  130°  C.  for 
two  to  ten  minutes.  (In  a  protracted  exposure  to  this  tem- 
perature, e.g.,  for  an  hour,  as  in  Ehrlich's  studies  of  the  gran- 
ules of  the  white  blood-cells,  the  bacteria  have  been  found  to 
lose  their  power  of  staining.)  This  heating  may  be  effected 
in  the  sterilizing  oven  (Fig.  1),  but  it  is  more  convenient  to 
follow  Koch  in  passing  the  cover  three  times  through  the 
flame  of  a  Bunsen  burner.  The  cover-glass  is  seized  with  the 
forceps  by  one  corner,  the  smeared  side  up,  and  passed  three 
times  through  a  vertical  circle  about  a  foot  in  diameter,  the 
clean  side  of  the  cover  being  brought  down  against  the  top  of 
the  flame  each  time.  The  best  rapidity  for  the  flaming,  upon 
which  success  depends,  is  reached  by  taking  about  three  sec- 
onds for  describing  the  three  circles. 

Of  the  various  ways  in  which  the  staining  fluid  and  the 
hardened  preparation  can  be  brought  together,  we  usually 
employ  that  of  allowing  the  cover-glass  to  float  upon  the  fluid, 
film  downward.  In  all  of  the  following  manipulations,  care 
must  be  taken  to  remember  which  side  of  the  cover  bears  the 
bacteria,  for  when  the  film  is  very  thin  it  may  be  difficult  to 
distinguish  it.  Sometimes  it  may  be  recognized  by  scratching 
upon  the  cover  with  a  sharp  pin,  the  irregularities  of  the 
smeared  side  being  felt,  or  the  scratches  seen. 

The  smeared  and  dried  cover-glasses  can  be  kept,  with  or 
without  hardening,  for  an  indefinite  time  before  staining,  being 
simply  laid  in  an  ordinary  cover-glass  box,  the  different  sets 
separated  by  labels  of  the  size  of  the  covers.  [For  clinical 
purposes,  it  is  convenient  to  use  a  small  oblong'  box,  a  couple 


Bacteriological    Technology.  145 

of  indies  long1,  with  a  rack  inside  like  a  slide-box.  Twenty  or 
more  clean  covers  are  easily  carried  in  such  a  box,  which  is 
small  enoug-h  to  be  slipped  into  the  vest  pocket  or  instrument- 
case,  so  that  they  are  always  ready  for  use  at  the  desk  or  bed- 
side.— W.  T.]  Two  opposite  corners  are  lifted  with  the  thumb 
and  forefing-er  of  the  rig-ht  hand,  the  cover  is  held  horizontal, 
and  allowed  to  fall  upon  the  fluid  from  a  heig-ht  of  several 
centimetres.  The  time  required  for  the  action  of  the  staining* 
fluid  varies  according-  to  circumstances  from  a  few  minutes 
to  an  entire  day.  Long-  staining-  at  the  temperature  of  the 
air  can  often  be  replaced  by  a  shorter  staining-  at  a  hig-her 
temperature  (Koch,  Loeffler),  e.g.,  No.  IV.,  p.  147,  infra. 
This  is  best  effected  by  setting-  the  watch-glass  of  staining- 
fluid  on  which  the  cover  floats,  upon  the  tin  stand  (Fig-.  72), 
and  heating-  it  with  a  small  flame  until  vapor  rises  freely  from 
the  surface.  The  lamp  is  then  removed  for  a  short  time,  after 
which  the  heating-  is  renewed,  and  this  is  repeated  several 
times  for  a  few  minutes,  or  long-er,  as  may  be  necessary. 
Sometimes  it  may  be  convenient  to  heat  the  fluid  until  it 
beg-ins  to  boil,  e.g.,  in  No.  IV. 

When  the  staining-,  or,  rather,  overstating-  of  the  prepara- 
tion is  finished,  the  latter  is  washed  (partly  decolorized  or 
"  differentiated  ").  This  is  effected  according-  to  circumstances 
by  the  use  of  one  or  other  of  the  fluids  enumerated  above  (cf. 
pp.  140  and  141),  which  is  then  removed  with  water  either  by 
rinsing-  under  the  faucet  or  with  a  wash-bottle,  or  by  moving- 
the  cover  back  and  forth  in  a  larg-e  dish  of  distilled  water. 
Washing-  out  the  excess  of  color  is  the  most  difficult  part  of 
staining-,  because  no  rules  can  be  given  as  to  the  length  of 
time  it  oug-ht  to  be  continued  in  each  case:  frequently  the 
rig-ht  time  is  only  learned  by  experiment.  When  it  has  been 
finished,  the  preparation  can  be  examined  at  once  in  distilled 
water  or  g-lycerin. 

It  should  also  be  observed  that  preparations  stained  with 
Bismarck  brown  or  vesuvin  (No.  2),  can  be  permanently 
mounted  in  g-lycerin,  while  this  substance  decolorizes  prepara- 
tions stained  with  the  other  aniline  colors.  On  the  other  hand, 
a  concentrated  solution  of  acetate  of  potassium  (1:2)  serves 
especially  well,  according-  to  Koch,  for  the  preservation  of  bac- 
teria stained  with  violet  or  fuchsin,  and,  it  may  be  added,  of 
unstained  bacteria;  but  it  should  not  be  used  for  those  stained 

brown. 

10 


1 46  Bacteriological    Technology. 

If  the  preparation  is  to  be  permanently  mounted  in  balsam, 
it  is  next  dried,  by  carefully  wiping-  off  the  clean  side  of  the 
cover  with  a  piece  of  soft  linen,  and  holding-  it  obliquely  with 
one  edge  upon  a  piece  of  filter-paper,  so  that  as  much  water 
as  possible  will  flow  off  or  be  absorbed  by  the  paper.  Any 
large  drops  remaining  on  the  film  are  carefully  removed  with 
filter-paper,  and  the  cover  is  allowed  to  become  air-dry.  To 
hasten  the  drying,  the  cover  may  be  pressed  between  folds  of 
filter-paper  (Ehrlich),  waved  back  and  forth  through  the  air, 
or  air  may  be  blown  over  it  by  means  of  a  rubber  bulb  fur- 
nished with  a  pointed  giass  tube  (Kuehne). 

The  dried  preparation  is  mounted  in  Canada  balsam.  It  is 
most  convenient  to  use  this  greatly  thinned  with  xylol,  and 
kept  at  hand  in  collapsible  tubes.  Turpentine  can  also  be  used 
for  this  purpose,  but  chloroform  [and  benzol]  are  to  be  avoided, 
as  they  remove  the  basic  aniline  colors.  Of  the  ethereal  oils, 
clove-oil  is  especially  apt  to  decolorize  the  bacteria,  so  that 
sections  should  rather  be  cleared  with  turpentine,  oil  of  berga- 
mot,  or  cedar-oil.  A  drop  of  this  is  applied  to  the  middle  of 
the  slide,  and  the  cover  inverted  upon  this,  spreading-  it  in  a 
thin  layer  by  its  weight.  Further  treatment  is  unnecessary, 
but  as  it  requires  a  long-  time  for  the  xylol-balsam  to  harden 
sufficiently  to  fix  the  cover  immovably,  it  is  convenient  to  seal 
the  preparation  by  means  of  a  filtered  alcoholic  solution  of 
shellac,  which  may  be  given  a  handsome  green  color  by  the 
addition  of  a  little  methyl  green.  Several  layers  of  this 
cement  are  painted  around  the  edge  of  the  cover. 

In  the  preliminary  examination  of  a  fluid  containing  bac- 
teria, a  less  complicated  method  is  used.  After  drying-  and 
flaming  the  cover-glass  preparation,  it  is  stained  by  a  diluted 
alcoholic  solution  (No.  3)  of  methylene  blue  or  fuchsin,  rinsed 
with  water,  and  examined  at  once  in  water,  or,  after  drying-, 
in  cedar-oil  (c/.  No.  1). 

METHODS  OF  STAINING  COVER-GLASS  PREPARATIONS. 

(I.)  Koch's  Original  Method  (1878).— The  dried  film  is 
stained  for  a  few  seconds,  or  a  little  longer,  with  a  solution 
of  methyl  violet  or  fuchsin  (a  few  drops  of  the  saturated  alco- 
holic solution  to  15  to  20  cc.  water),  washed  with  water  or 
acetate  of  potassium  (1:10),  dried,  and  mounted  in  balsam; 


Bacteriological    Technology.  147 

or  it  is  stained  with  vesuvin  dissolved  in  equal  parts  of  gly- 
cerin and  water,  and  washed  and  mounted  in  glycerin. 

(II.)  Kuehne's  Methylene-Blue  Method.— The  film,  after 
drying-  and  flaming,  is  stained  for  five  minutes  in  carbolic  blue 
(No.  6),  rinsed  in  water,  differentiated  by  laying  in  dilute  hy- 
drochloric acid  (No.  12)  for  a  few  seconds  or  a  little  longer 
according  to  the  thickness  of  the  film,  rinsed  a  moment  in 
lithium-water  (No.  14),  washed  under  the  water-jet  for  fifteen 
seconds,  dried— if  necessary  by  use  of  the  bulb  —  slightly 
warmed  over  the  flame,  cleared  in  xylol,  and  mounted  in 
balsam. 

(III.)  Gram's  Method. — The  flamed  film  is  treated  for  one 
to  three  minutes  with  aniline  gentian  violet  (No.  9),  laid  in  the 
iodine  solution  (No.  16)  for  an  equal  time,  washed  in  alcohol 
till  it  appears  completely  decolorized,  dried,  and  mounted  in 
balsam. 

(IV.)  Koch-Ehrlich-Weigert  Tubercle  Method.— The  dried 
and  flamed  film  is  heated  in  a  watch-glass  of  Weigert's  ani- 
line methyl  violet  (No.  8)  until  bubbles  begin  to  form  beneath 
the  cover-glass,  when  it  is  allowed  to  stand  five  minutes,  de- 
colorized in  a  tray  of  25-per-cent  nitric  acid,  in  which  it  is 
moved  back  and  forth  for  at  most  five  seconds,  immediately 
rinsed  in  60-per-cent  alcohol  (No.  17)  until  the  blue  color  dis- 
appears (usually  not  over  one  or  two  seconds),  counter-stained 
for  five  minutes  in  a  saturated  aqueous  solution  of  vesuvin, 
rinsed  in  water,  dried,  and  mounted  in  balsam. 

(Y.)  Ziehl-Neelsen  Tubercle  Method. — The  flamed  film  is 
floated  on  a  watch-glass  of  carbolic  fuchsin  (No.  7)  three  to 
eight  minutes  (with  heat),  decolorized  in  25-per-cent  nitric  or 
sulphuric  acid,  treated  with  60-per-cent  alcohol  until  only  a 
rosy  tint  remains,  the  acid  then  being  completely  washed  out 
in  a  large  quantity  of  water,  dried,  and  mounted  in  Canada 
balsam,  with  or  without  previous  clearing  in  xylol. 

[For  rapidly  staining  the  hardened  sputum  film,  the  fol- 
lowing commonly-used  method  leaves  little  to  be  desired :  The 
cover-glass  is  held  in  the  forceps  by  one  corner,  film  up,  a  large 
drop  of  carbolic  fuchsin  is  placed  on  it  so  as  to  entirely  cover 
the  upper  side,  and  it  is  heated  directly  over  the  flame  until 
it  steams  fully  or  even  boils,  care  being  taken  not  to  let  any 
part  become  dry.  If  necessary,  a  second  drop  of  the  staining 
fluid  is  added  as  the  first  evaporates,  and  the  heating  is  con- 


148  Bacteriological    Technology. 

tinued  for,  perhaps,  half  a  minute.  After  rinsing-  most  of  the 
staining1  fluid  off  under  the  faucet,  the  cover  is  dropped  into 
very  dilute  nitric  acid  until  the  red  color  changes  to  a  dull 
olive,  or  begins  to  disappear  entirely  (which  usually  requires 
very  few  seconds),  after  which  it  is  at  once  moved  about  in  a 
dish  containing1  a  considerable  quantity  of  alcohol  until  the 
dye,  which  has  resumed  its  original  red  color,  ceases  to  be 
given  off  in  clouds.  If  the  film  still  retains  any  noticeable 
color,  it  is  dipped  into  the  acid  once  more  and  again  washed  in 
alcohol,  after  which  it  is  counter  stained  with  methylene  blue, 
rinsed  in  water,  and  may  be  at  once  examined,  or  dried  and 
mounted  in  balsam.  By  this  method,  a  good  permanent  prep- 
aration may  be  had  under  the  microscope  within  five  minutes 
of  the  time  when  the  needle  is  first  dipped  into  the  sputum  for 
the  transfer  of  a  little  to  the  cover-glass. 

While  there  are  some  advantages  in  floating  cover-glass 
preparations  upon  the  staining  fluid,  as  the  author  recom- 
mends, it  is  more  common  to  keep  dilute  alcoholic  solutions 
(No.  4)  of  methylene  blue,  gentian  violet,  and  fuchsin,  as  well 
as  the  standard  alkaline  blue  (No.  10)  and  carbolic  fuchsin 
(No.  7)  in  2  oz.  wide-mouthed  bottles  closed  with  bored  corks 
through  which  dropping-tubes  pass  into  the  middle  of  the  bot- 
tle, so  that  a  drop  of  the  desired  staining-fluid  may  easily  be 
allowed  to  fall  upon  the  hardened  film,  heat  being  applied 
when  necessary  by  holding  the  cover  above  a  Bunsen  burner 
until  steam  begins  to  rise. — W.  T.] 

Staining  Spores  on  the  Cover-glass. — When  bacilli  which 
contain  spores  are  stained  by  the  above  methods,  the  spores 
remain  uncolored,  so  that  they  appear  as  colorless  spots  within 
the  strongly  colored  rods.  This  indisposition  of  the  spores  to 
receive  coloring  matters  can  be  overcome  in  various  ways, 
and  it  was  simultaneously  shown  by  Buchner  and  Hueppe 
that  a  prolonged  exposure  to  heat,  which  even  unfits  the  veg- 
etative cells  of  bacteria  for  staining  (cf.  p.  144,  Ehrlich),  ex- 
actly adapts  the  spores  to  the  reception  of  basic  aniline  colors 
in  aqueous  or  dilute  alcoholic  solutions. 

("VI.)  If,  instead  of  passing  the  cover-glass  through  the 
flame  three  times  to  harden  it  (p.  144),  it  is  flamed  ten  times, 
or  heated  fifteen  to  twenty  minutes  at  120°  to  180°  C.,  and 
then  stained  with  an  aqueous  solution  of  one  of  the  usual  basic 
colors,  the  spores  become  deeply  stained;  but  the  staining  of 


Bacteriological    Technology.  149 

the  spores  is  isolated,  because,  as  has  been  said,  the  rods  have 
lost  their  receptivity  for  the  dye. 

There  is,  however,  a  means  of  obtaining1  double-stained 
preparations,  in  which  the  spores  take  one  color,  the  rods, 
another:  namely,  by  an  extremely  intense  application  of  the 
Ehrlich  tubercle  method. 

(VII.)  The  cover-glass  film  is  hardened  in  the  flame  as 
usual  (three  times),  stained  for  an  hour  floating-  on  hot  aniline 
fuchsin  (p.  144),  rinsed  in  water,  decolorized  in  25  parts  hydro- 
chloric acid  and  75  of  alcohol,  and  counter-stained  in  a  satu- 
rated aqueous  solution  of  methylene  blue. 

There  is  a  great  difference  in  the  readiness  with  which  the 
spores  of  different  bacilli  may  be  stained.  [B.  Megatherium 
and  B.  subtilis  are  g-ood  species  to  practise  with,  B.  anthra- 
cis  is  among  the  more  difficult.  The  endospores  of  yeasts 
may  be  stained  by  the  same  methods,  decolorization  being1 
here  effected  by  alcohol  without  the  addition  of  an  acid. 
Ziehl's  carbolic  fuchsin,  heated  upon  the  cover,  as  in  tubercle 
staining,  gives  excellent  results,  but  it  must  be  renewed  sev- 
eral times  and  the  boiling-  correspondingly  prolonged. — W.  T.] 

STAINING  THE  FLAGELLA  OF  BACTERIA. 

In  the  case  of  certain  large  bacteria,  e.g.,  the  forms  of  Beg- 
giatoa  roseo-persinica  first  thoroughly  studied  by  Warming, 
— the  flagella  can  be  seen  by  aid  of  a  good  objective  without 
any  preparation  whatever.  In  the  case  of  smaller  forms,  they 
can  only  be  demonstrated  by  staining  them,  or  even  by  means 
of  staining  and  photography;  but  none  of  the  methods  so  far 
indicated  are  applicable  to  this  purpose.  Koch,  however,  suc- 
ceeded in  demonstrating  them  by  the  use  of  a  saturated  aque- 
ous solution  of  extract  of  logwood,  added  to  the  fluid  contain- 
ing the  bacteria.  His  permanent  preparations  were  made  as 
follows : 

(VIII.)  The  bacteria  are  dried  on  the  cover-glass,  stained 
with  an  aqueous  solution  of  logwood,  laid  in  dilute  chromic 
acid  (which  forms  a  dark  brown  combination  with  the  dye), 
dried,  and  mounted  in  balsam. 

Neuhaus  advises  the  replacement  of  chromic  acid  by  neu- 
tral sodium  bichromate,  prepared  by  adding1  5-per-cent  soda 
solution  drop  by  drop  to  dilute  chromic  acid.  He  recommends 


15°  Bacteriological    Technology. 

the   following-  as  the  most  certain   means   of    staining-  the 
fl  agella  : 

(IX.)  The  cover-glass  preparation  is  boiled  for  five  minutes 
upon  black  logwood  ink  ("  Kaisertinte "),  from  which  it  is 
placed  for  fifteen  minutes  in  dilute  neutral  bichromate  of 
sodium,  this  being-  repeated  several  times. 

STAINING  BACTERIA  IN  SECTIONS. 

When  it  is  only  necessary  to  demonstrate  the  presence  of 
bacteria  in  the  various  org-ans,  it  often  suffices  to  make  cover- 
glass  preparations  by  rubbing-  on  it  a  little  of  the  juices  from 
a  fresh  cut  surface;  but  a  more  exact  examination  of  the 
number  of  bacteria,  and  of  their  distribution  in  the  tissues, 
can  be  made  only  by  means  of  sections.  Before  Weigert  pro- 
vided methods  of  staining-  bacteria  in  situ,  no  means  of  dem- 
onstrating them  existed,  except  by  the  use  of  acids  and  alka- 
lies, to  which  they  show  great  resistance. 

(X.)  When  a  fresh  section,  or  one  hardened  in  alcohol,  is 
treated  with  strong-  acetic  acid  or  dilute  (2  per  cent)  solution 
of  caustic  potash  or  soda,  it  becomes  almost  transparent,  only 
the  bacteria  resisting  the  acid  or  alkali,  and  so  becoming  evi- 
dent, especially  when  collected  in  masses  in  or  without  the 
vessels;  hence  such  nests  of  cocci  were  seen  and  described 
long-  before  their  nature  was  known,  e.g.,  by  Beckmann,  in  the 
vessels  of  the  kidney.  Later,  the  potash  method  came  to  play 
an  important  part  in  the  study  of  pyaemia  early  in  the  seven- 
ties, e.g.,  in  the  work  of  Hj.  Heiberg;  and  quite  recently  it  has 
been  used  in  isolated  cases.  It  is  thus  used  to  render  collec- 
tions of  typhoid  bacilli  evident  in  the  tissues;  and,  even  before 
knowing-  of  Koch's  investigations  Baumgarten  had  seen 
tubercle  bacilli  by  the  use  of  the  potash  method.  Still,  in  gen- 
eral, this  has  lost  its  importance  since  the  introduction  of 
staining-  methods. 

One  condition  of  a  good  staining-  of  sections  is  their  being- 
well  and  completely  hardened.  The  organs  are  cut  into  small 
pieces  and  hardened  in  a  large  quantity  of  absolute  alcohol. 

[If  the  bottle  in  which  the  pieces  are  to  be  hardened  is 
filled  half  full  of  loose  cotton,  so  that  the  tissues  are  kept  near 
the  top  of  the  alcohol,  the  portion  of  this  which  becomes  more 
charged  with  water  tends  to  sink  to  the  bottom  because  of  its 


Bacteriological    Technology.  1 5 1 

greater  specific  gravity,  and  it  has  been  claimed  that  in  such 
cases  more  thorough  hardening-  is  effected. — W.  T.] 

As  thin  sections  as  possible  are  cut  from  the  well-hardened 
tissues  by  the  use  of  a  razor  or  microtome. 

[In  Koch's  laboratory  it  is  now  customary  to  use  a  drop  of 
melted  glycerin  -jelly  for  attaching  the  organ  to  a  cork,  by 
means  of  which  it  clamped  in  a  Schauze  or  other  sledge- 
microtome,  without  any  sort  of  imbedding.  If  perfectly  hard- 
ened material  is  used,  fairly  good  sections  are  obtained  in  this 
way,  the  sectioning  of  course  being  done  under  alcohol;  and 
there  is  no  danger  of  the  proper  staining-  of  the  bacteria  being- 
interfered  with.  But  in  many  cases,  where  the  organs  to  be 
sectioned  are  rather  large,  brittle,  etc.,  and  the  bacteria  will 
not  be  injured  by  the  preparatory  treatment,  the  tissue  may 
be  imbedded  in  celloidin  for  sectioning-  in  the  usual  way  under 
alcohol;  or  it  may  be  impregnated  with  paraffin  melting  at 
about  50°  C.,  and  imbedded  in  this  f or>  sectioning-  dry — prefer- 
ably with  some  form  of  rocking--microtome.  In  the  latter 
case,  if  the  org-an  is  small,  ribbon  sections  may  be  obtained 
and  stained  on  the  slide,  in  the  way  so  generally  employed 
now  by  histologists,  and  especially  embryologists;  or  the  par- 
affin may  be  removed  by  placing-  the  sections  in  turpentine 
for  a  few  moments,  and  afterward  in  alcohol,  after  which  they 
are  stained  individually  as  if  cut  without  imbedding. — W.  T.] 

The  staining  of  sections  is  essentially  the  same  as  staining 
cover-glass  preparations;  but  it  must  be  observed  that  as  a 
rule  they  require  a  longer  treatment  with  the  staining-fluid, 
are  less  tolerant  of  heating-  in  the  latter,  and  require  more 
powerful  decolorizing  media  for  differentiating  the  bacteria, 
while  anhydration  before  they  can  be  mounted  in  balsam  is 
rarely  effected  by  drying,  but  by  the  use  of  alcohol  or  anilin 
oil  (Weigert).  To  prevent  these  fluids  from  at  the  same  time 
partly  decolorizing-  the  preparation,  Kuehne  has  recently 
adopted  the  plan  of  anhydrating-  with  a  fluid  tinged  with  the 
same  dye  used  in  staining  the  bacteria  (No.  XIII.). 

Kuehne  advises  spreading  differentiated  sections  of  glanders 
material  upon  the  cover-glass,  blowing-  them  dry  (cf.  p.  146), 
laying-  the  cover-glass  (with  the  section  upward)  upon  a  giass 
plate,  where  it  is  slightly  warmed,  not  to  exceed  30°  C.,  over 
a  spirit  lamp,  until  the  section  becomes  transparent,  when, 
after  lying  on  the  warm  plate  for  five  minutes,  it  is  cleared 


152  Bacteriological    Technology. 

with  ethereal  oil  and  passed  through  xylol  into  balsam  [so 
that  the  use  of  alcohol  is  entirely  avoided  because  of  its 
marked  decolorizing-  action  on  the  bacteria]. 

For  the  microscopic  examination  of  sections  which  contain 
stained  bacteria,  a  sub-stage  condenser  is  used.  The  Abbe 
condenser  is  the  most  perfect  of  the  various  models  in  use, 
but  the  cheaper  Dujardin  and  similar  condensers,  which  are 
readily  used  on  smaller  stands  in  place  of  the  well-diaphragm, 
are  entirely  satisfactory. 

Koch  first  called  attention  to  the  importance  of  the  con- 
denser in  bacteriological  microscopy.  By  means  of  this  in- 
strument, as  he  expresses  it,  it  is  possible  to  efface  the  "struc- 
ture image  "  and  so  to  get  rid  of  its  disturbing  and  concealing 
influence  on  the  "color  image."  By  the  former  name  he 
designates  the  image  of  lines  and  shadows  which  reveals  to  us 
the  structure  of  the  tissue,  and  which  results  from  diffraction 
of  the  rays  of  light  during  their  passage  through  the  prepara- 
tion, different  parts  of  which  (nuclei,  fibres,  etc.)  differ  in  re- 
fractive power  from  the  substratum  in  which  they  lie  By  the 
"  color  image,"  he  refers  to  the  image  of  colored  parts,  which, 
if  very  small,  may  be  completely  concealed  by  the  lines  and 
shadows  of  the  structure  image.  By  the  use  of  an  Abbe  or 
similar  condenser  [with  full  opening],  the  preparation  is  illu- 
minated with  so  broad  a  cone  of  light  that  the  tissue  appears 
as  a  structureless  plane,  on  which  the  colored  parts,  large  and 
small,  stand  out  sharply.  It  is  easy  to  convince  one's  self  of  the 
great  advantage  of  using  such  a  condenser,  for  a  section  which 
is  full  of  stained  bacilli  of  mouse  septicaemia,  may  appear  free 
from  bacteria  when  examined  with  ordinary  illumination  with 
the  concave  mirror  and  diaphragm,  while  the  condenser  re- 
solves a  mass  of  colored  rods  within  it. 

What  is  here  said  -of  the  examination  of  stained  bacteria  in 
sections,  is  also  true  of  cover-glass  preparations,  in  which  the 
bacteria  frequently  occur  upon  or  within  elements  the  struc- 
ture images  of  which  mask  them.  On  the  other  hand,  it  must 
be  remembered  that  the  examination  of  unstained  bacteria 
should  always  be  made  without  a  condenser;  with  the  finest 
diaphragm  which  gives  sufficient  illumination;  or,  what 
amounts  to  the  same  thing,  with  the  condenser  lowered  as  far 
as  possible. 


Bacteriological    Technology.  153 


METHODS  OF  STAINING  SECTIONS. 

Several  of  the  numbered  methods  which  follow  have  already 
been  described  for  cover-glass  preparations,  but  are  repeated 
here  with  the  changes  necessary  for  sections. 

(XI.)  Weigert's  Original  Method  (1876). — The  section  is 
laid  for  a  rather  long-  time  in  a  quite  strong  solution  of 
methyl  violet,  washed  out  in  dilute  acetic  acid,  anhydrated  in 
alcohol,  cleared  in  oil  of  cloves,  and  mounted  in  balsam. 

Weigert's  improved  method  (1881)  employs  an  aqueous  1- 
per-cent  solution  of  gentian  violet,  BR.,  from  which  the  sec- 
tion g-oes  to  absolute  alcohol,  clove  oil,  and  balsam.  (Weigert 
recommended  gentian  violet,  BR.,  especially  because  it  is  not 
so  easily  removed  from  the  bacteria  when  the  section  is  treated 
with  alcohol  and  clove  oil.) 

(XII.)  Loefflers  Potash  Blue  Method. — The  sections  are 
placed  for  a  few  minutes  in  alkaline  methylene  blue  (No.  10), 
differentiated  for  some  seconds  in  0.5-per-cent  acetic  acid,  an- 
hydrated in  alcohol,  cleared  with  cedar-oil,  and  mounted  in 
balsam. 

(XIII.)  Kuehne  Universal  Method.8 — The  section  is  laid  in 
carbolic  methylene  blue  (No.  6),  as  a  g-eneral  thing-  for  half  an 
hour  (but  for  leprosy,  two  hours),  rinsed  with  distilled  water, 
treated  with  dilute  hydrochloric  acid  (No.  12)  until  the  color 
is  pale  blue,  rinsed  in  lithium-water  (No.  14),  laid  in  distilled 
water  for  some  minutes,  dipped  for  anhydration  into  absolute 
alcohol  (which  may  be  colored  with  methylene  blue,  p.  590), 
before  it  goes  into  a  salt-cellar  of  aniline-oil  colored  with 
methylene  blue,  where  in  a  few  moments  it  becomes  anhy- 
drated without  being-  decolorized,  then  rinsed  in  pure  aniline 
oil,  cleared  in  turpentine,  and  when  all  aniline  oil  has  been 
carefully  removed  by  treatment  with  xylol,  usually  renewed 
once,  it  is  mounted  in  balsam.  When  the  aniline  oil  is  not 
completely  removed,  the  balsam  becomes  brown  with  time. 

The  differentiation  in  hydrochloric  acid  is  the  most  difficult 
point  in  the  process.  Thin  sections  need  only  be  dipped  into 
it;  and  in  any  case  a  sharp  watch  must  be  kept  upon  the  de- 
gree to  which  decolorization  has  progressed,  so  that  the  sec- 
tion may  be  rinsed  in  lithium  water  at  once  when  the  right 
shade  is  reached. 


154  Bacteriological    Technology 

(XIV.)  Koch's  Isolated  Staining  Method. — After  staining 
in  an  aqueous  solution  of  fuchsin,  methyl  violet,  or  methylene 
blue,  the  sections  are  washed  out  in  a  half  saturated  solution 
of  carbonate  of  potassium  (No.  15  a),  then  passed  through 
alcohol,  xylol,  and  balsam.  Weigert  was  the  first  to  undertake 
an  isolated  staining  of  bacteria  in  tissues,  since  by  the  use  of 
carmine,  followed  by  washing  in  glycerin  acidulated  with  hy- 
drochloric acid,  he  stained  clusters  of  micrococci  in  1871. 

Malassez  and  Vignal,  after  staining  in  methylene  blue, 
wash  in  No.  15  b,  etc. 

(XV.)  The  Grain  Method. — The  sections  are  transferred 
directly  from  absolute  alcohol  -into  aniline  gentian  violet,  and 
treated  like  cover-glass  preparations  (No.  III.),  except  that 
they  are  anhydrated  in  absolute  alcohol  and  cleared  in  cedar- 
oil  preparatory  to  being  mounted  in  balsam.  For  Weigert's 
modification  of  the  method,  see  No.  XIX.,  infra. 

When  sections  stained  by  this  method  are  also  treated  with 
a  nuclear  stain  like  carmine,  a  very  pretty  double  staining  is 
obtained.  Fraenkel  recommends  the  following  method,  which 
is  also  thought  to  more  frequently  escape  the  troublesome 
granular  precipitates  which  now  and  then  appear  in  the  prep- 
aration when  the  original  Gram  method  is  used. 

(!XVI.)  The  sections  are  transferred  from  alcohol  to  strong 
picro-carmine,  where  they  remain  half  an  hour,  are  rinsed  in 
50-per-cent  alcohol,  then  go  for  half  an  hour  into  unsaturated 
aniline  gentian  violet — prepared  by  dropping  a  few  drops  of 
the  saturated  alcoholic  solution  (No.  1)  into  a  watch-glass 
containing  aniline  wrater  (No.  5)  until  the  mixture  begins  to  be 
opaque — from  which  they  are  placed  directly  in  the  iodine 
water  for  three  minutes,  then  into  alcohol,  where  the  red  color 
reappears  and  they  become  anhydrated,  when  they  go  through 
cedar-oil  into  balsam. 

(XVII.)  Koch-Ehrlich-Weigert's  Tubercle  Method.— The 
sections  are  laid  in  aniline  gentian  violet  (No.  9)  for  twelve 
hours,  moved  about  for  a  few  seconds  in  25-per-cent  nitric  acid, 
rinsed  in  60-per-cent  alcohol  until  they  chow  only  a  slight  blue 
color,  counter-stained  for  some  minutes  in  a  saturated  aqueous 
solution  of  vesuvin,  rinsed  in  60-per-cent  alcohol,  anhydrated 
in  absolute  alcohol,  cleared  with  cedar-oil,  and  mounted  in 
xylol  balsam. 

The  tubercle  bacilli  are  also  stained  by  Kuehne's  method 


Bacteriological    Technology.  155 

(XIII.)  and  the  Gram  method,  but  where  a  differential  diag- 
nosis is  required  decolorization  must  be  effected  by  strong* 
mineral  acids  (e.g.,  as  in  Nos.  XVII.  and  XVIII.) ;  for  it  is  its 
resistance  to  decolorization  by  mineral  acids  which  distin- 
guishes the  tubercle  bacillus  from  all  other  known  species  ex- 
cept the  bacillus  of  leprosy,  which,  however,  differs  from  it  in 
staining  readily  with  a  simple  aqueous  solution  of  methyl 
violet  or  fuchsin.  The  so-called  smegma-bacilli  also  show  a 
decided  power  to  resist  the  action  of  mineral  acids,  but  they 
are  readily  decolorized  by  subsequent  treatment  with  alcohol, 
which  is  not  the  case  with  either  the  tubercle  or  leprosy  bacil- 
lus. The  smegma-bacilli  are  probably  only  different  species  of 
putrefactive  bacteria  which,  from  growing  in  the  smegma> 
have  become  so  impregnated  with  oily  matters  that  they  have 
become  difficult  to  stain.  This  view  is  supported  by  the  fact 
that  it  has  proved  possible  to  change  the  behavior  of  other 
species  toward  staining  methods,  by  growing  them  in  sub- 
stances containing  butter,  etc.  (Bienstock).  On  the  other 
hand,  the  diagnosis  of  smegma-baciUi  may  be  attended  with 
some  difficulty  as  compared  with  Lustgarten's  syphilis  bacil- 
lus, the  etiological  relation  of  which  to  the  disease  is  still 
very  problematical,  although  it  has  been  demonstrated  in 
syphilitic  secretions  and  neoplastic  growths.  Like  the  syphilis 
bacillus,  after  being  stained  on  the  cover-glass  for  twenty -four 
hours  in  aniline  gentian  violet,  they  remain  colored  after  treat- 
ment for  ten  seconds  with  1.5-per-cent  solution  of  potassium 
permanganate,  followed  by  sufficient  washing  in  sulphurous- 
acid  water  to  entirely  decolorize  the  preparation.  The  syph- 
ilis bacillus  cannot  be  confounded  with  that  of  tubercle,  be- 
cause it  is  very  easily  decolorized  by  mineral  acids. 

(XVIII.)  Ziehl-Neelsen  Tubercle  Method.— Sections  are 
more  rapidly  stained  by  laying  them  for  fifteen  minutes  in 
carbolic  fuchsin  (No.  7),  agitating  them  a  few  seconds  in  25 -per- 
cent sulphuric  or  nitric  acid,  rinsing  in  60-per-cent  alcohol 
until  only  a  light  rosy  color  remains,  counter-staining  for  some 
minutes  in  a  saturated  aqueous  solution  of  methylene  blue,  and 
rinsing,  anhydrating,  and  mounting  as  before. 

For  staining  bacteria  in  sections  of  hardened  gelatin-cul- 
tures, see  Neisser,  "Centralbl.  f.  Bakteriologie,"  1888,  III.,  506. 


156  Bacteriological    Technology. 


MOULDS. 

The  following  notes  on  mounting-  moulds  refer  chiefly  to 
the  directions  of  O.  Johan-Olsen. 

Temporary  preparations  are  obtained  by  laying  the  mould 
for  a  few  minutes  in  dilute  alcohol,  then  in  weak  ammonia, 
which  is  removed  with  filter-paper,  the  surplus  washed  out 
with  water,  and  this  replaced  by  glycerin,  in  which  the  exam- 
ination is  made. 

Permanent  preparations  are  made  in  the  same  manner, 
after  staining  the  material  in  osmic  acid,  which  is  kept  for  use 
in  a  0.5-per-cent  solution  in  a  dropping -bottle  that  has  been 
well  cleansed  with  alcohol  and  ether  and  is  stopped  with  glass. 
Two  drops  of  this  are  added  to  eight  drops  of  water  in  a 
watch-glass  (i.e.,  about  0.1  per  cent),  in  which  the  mould  is 
allowed  to  lie  for  an  entire  day;  or,  if  the  0.5-per-cent  solution 
is  used  directly,  a  few  minutes  suffice.  The  preparation  is 
then  washed  with  alcohol,  followed  by  distilled  water.  If  it  is 
still  too  black,  it  is  decolorized  sufficiently  in  weak  ammonia, 
followed  by  water,  and  mounted  in  glycerin.  Preparations 
stained  with  osmic  acid  may  also  be  further  stained  by  saffra- 
nin,  usually  a  very  dilute  solution,  in  which  they  are  allowed 
to  remain  for  a  long  time. 

Preparations  of  an  entire  culture  can  be  made  upon  the 
slide.  Usually  the  cultures  are  obtained  from  fluid  media 
(e.g.}  Brefeld's  slide-cultures,  supra,  p.  100),  and  those  which 
are  not  too  far  advanced  are  chosen.  The  colony  being  placed 
on  a  slide,  a  cover-glass  is  let  down  upon  it  carefully — espe- 
cially in  case  of  the  erect  forms — and  it  is  allowed  to  dry 
down  so  that  the  cover -glass  does  not  move  around  (usually 
only  a  few  minutes  being  needed),  when  0.1-per-cent  osmic  acid 
is  added  at  one  side  and  drawn  under  by  applying  bibulous 
paper  to  the  other  edge  of  the  cover.  After  a  proper  time 
the  acid  is  thoroughly  washed  out  with  water  that  is  drawn 
under  the  cover  in  the  same  way,  and,  either  with  or  without 
saffranin  staining,  mounted  by  drawing  glycerin  under  and 
sealing  in  the  usual  way:  or  else  dilute  alcohol  is  used  instead 
of  the  osmic  acid,  and  followed  by  ammonia,  etc.,  as  indicated 
above  for  temporary  preparations. 

For  demonstrating  moulds  in  sections,  Hueppe  recommends 


Bacteriological    Technology.  157 

the  methylene  blue  universal  method  (Nos.  XII.  and  XIII.). 
Blbbert  advises  the  following1  modification  of  the  Gram 
method : 

(XIX.)  Weigert's  Fibrin  Stain. — The  section  hardened  in 
alcohol  is  stained  for  a  long-  time  in  saturated  aniline  gentian 
violet  (No.  9),  rinsed  in  0.7-per-cent  solution  ©f  table  salt,  and 
transferred  to  the  slide,  where  the  other  steps  are  taken.  The 
superfluous  water  is  removed  with  filter-paper,  iodine  water 
(No.  16)  is  dropped  upon  it  and  removed  with  filter  paper,  and 
a  couple  of  drops  of  aniline  oil  allowed  to  fall  upon  the  section 
for  the  purpose  of  at  once  decolorizing  and  anhydrating  it. 

As  the  aniline  oil  soon  becomes  dark,  it  must  be  removed 
and  replaced  by  fresh  oil  once  or  twice,  after  which  it  is  en- 
tirely replaced  by  xylol  and  the  section  mounted  in  balsam. 


ACTINOMYCES. 

When  it  is  only  desired  to  obtain  a  sure  diagnosis  of  Acti- 
nomyces  cushions  in  pus  or  sections,  treatment  with  acetic 
acid  or  alkalies  and  examination  in  glycerin  suffices.  For 
staining  cover-glass  preparations,  Fraenkel  employs  the  Gram 
method,  letting  the  section  stay  in  aniline  gentian  violet  for 
twenty -four  hours,  and  in  the  iodine  solution  for  fifteen  min- 
utes. The  Weigert-Gram  method  (No.  XIX.)  appears  suited 
to  staining  sections  for  Actinomyces. 

AMCEBOIDS. 

The  amoebae  or  amoeboid  organism  detected  by  Laveran 
and  subsequently  more  fully  studied  by  Marchiafava  and  Oelli, 
Golgi,  Osier,  and  others,  and  held  to  be  the  cause  of  malaria, 
are  not  adapted  to  staining  by  any  of  the  above  methods.  It 
is,  indeed,  possible,  when  cover-glass  preparations  of  the  blood 
are  treated  with  methylene  blue  or  vesuvin,  to  find  within  the 
red  corpuscles  larger  or  smaller  irregular  spots,  colored  blue 
or  brown  (Marchiafava  and  Celli).  These  are  the  stained 
amoebae;  but  a  clear  view  of  all  the  developmental  forms  of 
the  parasite  is  obtained  only  by  examination  of  the  fresh 
blood.  Laveran  gives  the  following  directions  for  this :  The 
amoebae  are  most  easily  found  in  patients  already  anaemic,  and 
who  have  not  taken  quinine,  at  any  rate  just  before  the  exam- 


158  Bacteriological    Technology. 

ination.  The  blood  is  obtained  just  before,  or  :n  the  beginning 
of  an  attack,  by  a  needle  puncture  in  the  finger-tip  or  lobe  of 
the  ear,  after  careful  cleansing-  with  water  and  alcohol.  The 
surface  of  the  drop  which  exudes  is  touched  with  a  well- 
cleaned  slide,  and  the  small  drop  of  blood  which  adheres  is 
quicidy  covered  with  a  cover-glass,  and  sealed  with  paraffin 
when  the  layer  of  blood  is  thin  enough  for  examination.  An 
enlargement  of  400  to  500  diameters  is  sufficient  for  the  inves- 
tigation, places  being  chosen  where  the  red  corpuscles  lie  flat 
and  isolated.  Few  parasites  are  usually  to  be  seen,  rarely  as 
many  as  eight  to  fifteen  in  the  same  field,  so  that  one  must  be 
prepared  to  spend  some  time  in  searching-  for  them.  The  pig- 
ment granules  that  occur  in  most  of  the  amoebae  give  a  clue 
in  the  search.  Richard  recommends  the  addition  of  a  drop  of 
dilute  acetic  acid  for  the  rapid  demonstration  of  these  para- 
sites, as  it  removes  the  annoying-  red  blood-corpuscles  without 
destroying  the  parasites.  According  to  Laveran,  the  addition 
of  water  is  better,  as  it  does  not  kill  the  amoebae  so  soon  as 
acetic  acid  does,  but  allows  them  to  continue  their  peculiar 
movements. 


BIBLIOGRAPHY. 

1.  Fortschritt.  d.  Medicin,  1886,  iv.— 2.  Meddelelser  f.  Carlsberg 
Lab.,  ii.,  218.— 3.  C/.  Fortsch'r.  d.  Med.,  1888,  No.  1.— 4.  Zeitschr.  f.  Hy- 
giene, 1887,  521.— 5.  Ann.  de  Micrographie,  i.,  153. — 6.  Sur  la  culture  des 
microbes  anae*robies  (Annales  de  1'Institut  Pasteur,  1887,  No.  2).— 
7.  Beitrage  zur  Kenntniss  des  Sauerstoffbediirfnisses  der  Bacterien 
<Zeitschr.  fur  Hygiene,  1886,  i.,  115).— 8.  FromKuehne:  Praktische  An- 
leitung  zum  mikroskopischen  Nachweis  der  Bakterien  iin  thierischen 
Gewebe,  Leipzig. 


INDEX. 


ACID,  pyrogallic,   removal  of  oxy- 
gen by,  92. 

Actinomyces,  examination  of,  157. 
Aeroscope,  Hesse's,  71. 
Miquel's,  73. 
Schoenauer's,  68. 
Straus  and  Wurtz's,  74. 
Agar-agar  for  culture,  25. 
for  plate  culture,  62. 
peptonized,  preparation  of,  28. 
Agar-gelatin  for  culture,  26. 
for  plate  culture,  63. 
peptonized,  preparation  of,  28. 
Air,  bacteriological  analysis  of,  66. 
Air-pump,  cultivation  of  anaerobic 

bacteria  by  the  aid  of  the,  87. 
Alg'se,  use  of,  in  examination  of  bac- 
teria, 133. 

Amoeboids,  examination  of,  157. 
Anaerobic  bacteria,  cultivation  by 

the  aid  of  the  air-pump,  87. 
bacteria,  culture  of,  80. 
bacteria,  culture  vessels  for,  88. 
bacteria,  isolation  by  the  capilla- 
ry-tube method,  86. 
bacteria,  isolation  in  gelatinized 

media,  85. 

bacteria,  isolation  of,  83. 
bacteria,  roll-culture  of,  87. 
culture  by    the  aid   of  aerobic 

bacteria,  91. 
culture  in  hermetically  sealed 

tubes,  90. 

culture,  pipettes  for,  91. 
culture,  preservation  of,  90. 
culture    under    a    gelatinizing 

plug,  90. 

culture  under  oil,  90. 
Analysis,  bacteriological,  of  various 

substances,  55. 
Aniline  staining,  134. 
Animals,  inoculation  of,  102. 
Apparatus,  bubbling,  for  air  analy- 
sis, 72. 

for  culture,  14. 
Arachnoid,  inoculation  beneath  the, 

110. 
Aspirators  for  air  analysis,  66. 


BACILLUS  anthracis,  effect  of  car- 
bolic acid  on,  132. 
Bacteria,  anaerobic,  cultivation  by 

the  aid  of  the  air-pump,  87. 
anaerobic,  culture  of,  80. 
anaerobic,  culture  vessels  for,88. 
anaerobic,  isolation  by  the  capil- 
lary-tube method,  86. 
anaerobic,  isolation  in  gelatin- 
ized media,  85. 
anaerobic,  isolation  of,  83. 
anaerobic,  roll-culture  of,  87. 
in  sections,  staining,  150. 
microscopic  examination  of,  133. 
staining  flagella  of,  149. 
staining*  of,  133. 
staining  of,  in  fluids,  142. 
Bacteriological  analysis   of  various 

substances,  55. 
Baumgarten,  staining    method    of, 

135. 

Beer-wort  for  culture,  20,  29. 
Bench  of  sheet  zinc  for  supporting 

a  plate  culture,  60. 
Blood,  collection  of  sterile,  31,  119. 

serum,  sterilization  of,  8. 
Boettcher's  moist  chamber,  97. 
Bohr's  thermo-regulator,  51. 
Bottles  for  culture,  15. 
Bouillon  for  culture,  19. 
Box,  glass,  for  culture,  17. 
Bread,  white,  for  culture,  prepara- 
tion of,  32. 
Brood-ovens,  48. 

Bubbling  apparatus  for  air  analysis, 
72. 

CAPILLARY  tubes   for    inoculation, 
44,  45. 

Capillary -tube  method    of    separa- 
tion, 39. 

Cement     for     fastening     capillary 
tubes,  40. 

Chamber,  Geissler,  99. 
moist,  Boettcher's,  97. 
moist,  Ranvier's,  98. 

Chamberland  flasks,  17. 

Chambeiiand's  filter,  9. 


i6o 


Index. 


Chamberland's  filter,  cleaning' of,  12. 
Chambers,  moist,  94. 
Cibil's  extract  for  culture,  20. 
gelatin,  preparation  of,  27. 
Clarifying  of  nutrient  jelly,  26. 

preparations,  146. 

Cleanliness    in   sterilization,    neces- 
sity for,  7. 

Cohn's  heating-  method   for  obtain- 
ing pure  cultures,  38. 
Condenser,  necessity  for  use  of,  in 

examination  of  sections,  152. 
Contamination     of     sterilized    ob- 
jects, guarding  against,  4. 
Cooling    apparatus    for    plate    cul- 
tures, 59. 

Counting  germs,  41. 
Cracker  box  for  sterilization,  2. 
Cultivation  of  micro-organisms  un- 
der the  microscope,  94. 
Culture,   anaerobic,    by  the  aid  of 

aerobic  bacteria,  91. 
anaerobic,  in  hermetically  sealed 

tubes,  90. 

anaerobic,  pipettes  for,  91. 
anaerobic,  preservation  of,  90. 
anaerobic,  under  a  gelatinizing 

plug,  90. 

anaerobic,  under  oil,  90. 
apparatus,  14. 
bottles  for,  15. 
flasks  for,  15. 
glass  box  for,  17. 
glasses,  15. 
media,   and  their   introduction 

into  vessels,  18. 

media,  collection  of  sterile,  118. 
media,  preparation  of,  18. 
obtaining  pure  material  for,  36. 
of  anaerobic  bacteria,  80. 
potato,  in  a  vacuum,  92. 
test-tubes  for,  14. 
vessels,  tilling  of,  34. 
Cultures,  collection  of  dust  for,  69. 
from  man  and  animals,  115. 
inoculation  of,  43. 
Klebs'  fractional,  37. 
pure,   Cohn's   heating    method 
for,  38. 

DECOCTION  of  dried  fruits  for  cul- 
ture, 20,  29. 

of  horse  dung  for  culture,  20. 
of  liver,  etc.,  for  culture,  20. 
of  prunes  for  culture,  20. 
of  wheat,  hay,  cabbage,  for  cul- 
ture, 20. 
Digestive   tract,   infection   through 

the,  112. 

Dilution,  method  of,  for  obtaining 
pure  cultures,  41. 


Discontinuous  heating,  sterilization 

by,  7. 

Disinfectant,  testing  a  fluid,  126. 
Disinfectants  in  sterilization,  13. 
Disinfecting  oven,  diagram  of,  130. 

oven,  testing  a,  128. 
Disinfection  experiments,  121. 
Drip-aspirator,  67. 
Drying-jar  for  hydrophobia  vaccine, 

Dust,  collection  of,  for  cultures,  69. 
collection  of,  for  microscopical 
examination,  67. 

EGGS  for  culture,  preparation  of,  33. 
Erlenmeyer  flasks,  15,  18. 
Extracts  of  meat  for  culture,  20. 
Eye,    inoculation    of    the    anterior 
chamber  of  the,  109. 

FIBRIN  stain,  Weigert's,  157. 
Filling  culture  vessels,  34. 
Filter,  Chamberland's,  9. 

Chamberland's,  cleaning  of,  12. 

simple,  for  gelatin,  27. 
Filtering  of  nutrient  jelly,  26. 
Filters,  insoluble  powder,  74. 

porcelain,  9. 

sand,  74. 

soluble,  75. 
Filtration,  10. 
Finder,  simple,  97. 
Flagella  of  bacteria,  staining  of,  149. 
Flask,  Pasteur-Chamberland,  17. 
Flasks,   conical,    in    bacteriological 
analysis,  56. 

for  culture,  15. 
Flesh  water  for  culture,  18,  19. 

GAG  for  use  in  feeding  experiments, 

112. 

Geissler  chamber,  99. 
Gelatin,  Cibil's,  preparation  of,  27. 
colored    nutrient,   for    culture, 

preparation  of,  33. 
filter  for,  27. 
for  culture,  25. 
for  plate  culture,  62. 
Gelatinized    media,   separation    of 

germs  by,  42,  56,  58. 
Germs,  counting,  41. 
Glass  box  for  culture,  17. 

needles  for  inoculation,  45. 
plates  in  bacteriological  analy- 
sis, 58. 

trays  for  culture,  24. 
trays  in  bacteriological  analysis, 

57. 

Glycerin  B.  P.  A.  for  culture.  28. 
Glycerin-serum,  32. 
Gram's  staining  method,  147,  154. 


Index. 


161 


HAY  infusion,  sterilization  of,  7. 
Heat,  sterilization  by,  1. 
Heating,     discontinuous,     steriliza- 
tion by,  7. 

Hesse's  aeroscope,  71. 
Hydrogen   generator,   Joergensen's 

83. 
Pasteur     pipette    for    passing, 

through  gelatin,  87. 
Hydrophobia     vaccine,     drying- jar 

for,  112. 
virus,  110. 

INDIGOTIN  test  for  oxygen,  82. 
Infection  by  inhalation,  113. 

through  the  digestive  tract,  112. 

through  the  trachea,  113. 
Infusion  of  wheat,  hay,  cabbage,  for 

culture,  20. 

Inhalation,  infection  by,  113. 
Inoculation,  capillary  tubes  for,  44, 
45. 

glass  needles' for,  45. 

of  a  test-tube,  46. 

of  animals,  102. 

of  cultures,  43. 

of  the  anterior  chamber  of  the 
eye,  109. 

Pasteur  pipettes  for,  44,  46. 
Irish -moss  for  culture,  26. 

JELLY,  nutrient,  clarifying  of,  26. 

nutrient,  filtering  of,  26. 
Joergensen's    hydrogen    generator, 

83. 

KLEBS'  fractional  cultures,  37. 
Knife-rests  for  supporting  pipettes, 

35. 

Koch-Ehrlich-Weigert's  staining 
.  method,  147,  154. 
Koch's  method  of  separating  germs, 
42,  56,  58. 

staining  method,  143,  146,  154. 

steam  sterilizer,  4. 
Kuehne's  staining  method,  147,  153. 

LEVELLING  tripod,  60. 

Liborius'  method  of  isolation  of  bac- 
teria, 84. 

Liebig's  extract  for  culture,  20. 

Liquids,  bacteriological  analysis  of, 
56. 

Loeffler's  staining  method,  153. 

MATERIAL    for    culture,   obtaining 

pure,  36. 

Meat  broth  for  culture,  18. 
Media,  fluid,  for  culture,  18. 
solid,  for  culture,  21. 


Mica,  plate  cultures  under,  84. 
Mice,  keeping*,  103. 
Microscope,   cultivation    of    micro- 
organisms under  the,  94. 
Microscopic  examination  of  bacte- 
ria, 133. 
Miquel's  aeroscope,  73. 

soluble  powder  filter,,  76. 
Moist  chamber,  Boettcher's,  97. 

chamber,  Ranvier's,  98. 

chambers,  94. 
Moulds,  culture  media  for,  20,  29. 

mounting,  156. 
Mounting  moulds,  156. 

preparations,  146. 
Mouse  jar,  103. 

OVEN,  sterilizing,  2. 

Oxygen,  indigotin  test  for,  82. 

removal  of,  by  pyrogallic  acid, 
92. 

PAPIN   digester  for  sterilization,  2, 

7. 
Paraffin,   application   of,   to  slides, 

133. 

Pasteur  culture  vessels,  89. 
flasks,  17. 
pipette  with  rubber  cap  plugged 

with  cotton,  119. 
pipettes  for  inoculation,  44,  46. 
wash-bottle,  118. 
Pasteurization,  8. 
Petri's  sand  filter,  75. 
Physiological  differences,  utilization 
of,  in  obtaining  pure  cultures,  37. 
Pipette  with  cotton  plug,  34. 
Pipettes  for  anaerobic  culture,  91. 
Pasteur,  for  inoculation,  44,  46. 
Pasteur,      with      rubber      cap 

plugged  with  cotton,  119. 
Plan  tarn  our  hot- water  funnel  for  fil- 
tration of  gelatin,  28. 
Plaster,  moist,  for  culture,  prepara- 
tion of,  33. 

Plate  culture,  agar-agar  for,  62. 
culture,  agar-gelatiri  for,  63. 
culture,  bench  of  sheet  zinc  for 

supporting,  60. 
culture,  gelatin  for,  62. 
culture,  serum  for,  63. 
culture,  Soyka's  method,  61. 
cultures,  cooling  apparatus  for, 

59. 

cultures,  preparation  of,  59. 
cultures  under  mica,  84. 
Platinum  needle  for  inoculation,  43, 
45. 


Plugs,  tubular,  16. 
Porcelain  filters,  9. 


162 


Index. 


Potato,  boiled,  for  culture,  23. 
broth  for  culture,  25. 
cultures  in  a  vacuum,  92. 
Powder  filters,  insoluble,  74. 
Purification  blotches  in   cylindrical 

glass,  40. 

Pyrogailic  acid,  removal  of  oxygen 
by,  92. 

RABBITS,  inoculation  of,  107. 
Rabies,  inoculation  of,  110. 

vaccines,  preparation  of,  111. 
Ranvier's  moist  chamber,  98. 
Rei chert's  thermo-regulator,  51. 
Rice    milk  for  culture,  preparation 

of,  33. 

Rohrbeck's  thermo-regulator,  51. 
Roll-culture  of  anaerobic  bacteria, 

87. 

SAND  filters,  74. 
Schoenauer's  aeroscope,  68. 
Sections,  staining  bacteria  in,  150. 
Separation,  methods  for  obtaining 

pure  cultures  by,  39. 
Serum  for  culture,  preparation  of, 
29. 

for  plate  culture,  63. 

of  blood,  sterilization  of,  8. 
Slide,  hollow-ground,  95. 
Soil,  artificial,  for  culture,  prepara- 
tion of,  33. 

bacteriological  analysis  of,  64. 
Solids,  bacteriological   analysis   of, 

64. 

Soluble  filters,  75. 
Soyka's  box,  17. 

method  of  plate  culture,  61. 
Spores,  staining,  on  the  cover  glass, 

148. 

Sputum,  staining,  147. 
Stain,  Weigert's  fibrin,  157. 
Staining  bacteria,  133. 

bacteria  in  fluids,  142. 

bacteria  in  sections,  150. 

differential,  136. 

double,  137. 

flagella  of  bacteria,  149. 

materials  employed  in,  139. 

method,  Baumgarten's,  135. 

method,  Gram's,  147,  154. 

method,  Koch's,  143,  146,  154. 

method,  K  o  c  h-E  h  r  1  i  c  h-Wei- 
gert's,  147,  154. 

method,  Kuehne's,  147,  153. 

method,  Loeffler's,  153. 

method,  Weigert's,  153. 

method,    Ziehl-Neelsen's,     147, 
155. 

methods  of,  146. 

mordants  used  in,  140. 


Staining  preparations  dried  on  the 
cover  glass,  143. 

sections,  methods  of,  153. 

spores  on  the  cover  glass,  148. 

sputum,  147. 

Steam  sterilizer,  Koch's,  4. 
Sterilization,  1. 

by  discontinuous  heating,  7. 

by  heat,  1. 

by  steam,  4. 

cracker  box  for,  2. 

disinfectants  in,  13. 

instruments  needed  for,  2. 

of  blood  serum,  8. 

of  hay  infusion,  7. 

Papin  digester  for,  2,  7. 

water  bath  for,  8. 
Sterilizer,  Koch's  steam,  4. 
Sterilizing  oven,  2. 
Stoppers,  tubular,  16. 
Straus  and  Wurtz's  aeroscope,  74. 
Suction-bulb,  automatic,  for  collect- 
ing air  germs,  70. 
Support  for  funnels,  etc.,  142. 

TESTING  a  disinfecting  oven,  128. 

a  fluid  disinfectant,  126. 
Test-tube,  inoculation  of  a,  46. 
Test-tubes  for  culture,  14. 

in  bacteriological  analysis,  56. 

plugging  of,  14. 
Thermometer,  electric,  128. 
Thermo-regulators,  48. 
Thermostat   for   solidifying  serum, 

31 

Thermostats,  49. 
Tripod,  levelling,  60. 
Tubular  plugs,  16. 

VACCINE,    hydrophobia,    drying-jar 

for,  112. 

Vaccines,  rabies,  preparation  of ,  111. 
Vacuum  cultures  for  bacteria,  89. 

potato  culture  in  a,  92. 
Virus,  hydrophobia,  110. 

WAITING  for  germination  after  ster- 
ilization, 8. 

Wash-bottle,  Pasteur,  118. 
Water,  bacteriological  analysis   of, 

63. 

bath  for  sterilization,  8. 
Weigert's  fibrin  stain,  157. 
staining  method,  153. 
Wine  for  culture,  20. 

YEAST,  culture  media  for,  20,  29. 
water  for  culture,  20. 

ZIEHL-NEELSEN'S  staining  method, 
147,  155. 


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